Mutant alpha-synuclein, and methods using same

ABSTRACT

The present invention relates to a mutant human alpha-synuclein with increased toxicity compared to wild-type alpha-synuclein, or a homologue thereof, wherein the mutant alpha-synuclein or homologue thereof comprises at least one amino acid substitution selected from the group consisting of a substitution at the alanine at position 56 (A56), at the alanine at position 76 (A76), at the methionine at position 127 (M127) and/or at the valine at position 118 (V118), as defined in the claims. Further, the invention relates to a polynucleotide encoding the mutant alpha-synuclein or homologue thereof, or an expression vector comprising said polynucleotide, a cell comprising the polynucleotide or expression vector, as defined in the claims. Also, a non-human animal comprising the cell of the invention is provided, as defined in the claims. Finally, the invention provides methods for identifying a substance that prevents or reduces toxicity of alpha-synuclein, as defined in the claims.

This application is a Divisional application of U.S. patent applicationSer. No. 13/057,680, filed Aug. 12, 2011, which is a National Stageapplication of International Application No. PCT/EP2009/060299 filedAug. 7, 2009, the entire contents of which is hereby incorporated hereinby reference. This application also claims priority under 35 U.S.C. §119to European Patent Application No. 08162056.9, filed Aug. 8, 2008, theentire contents of which is hereby incorporated herein by reference.

The invention relates to mutant alpha-synuclein with increased toxicitycompared to wildtype alpha-synuclein. In particular, the inventionrelates to a mutant human alpha-synuclein, or a homologue thereof,comprising at least one amino acid substitution selected from the groupconsisting of a substitution at the alanine at position 56 (A56), at thealanine at position 76 (A76), at the methionine at position 127 (M127)and/or at the valine at position 118 (V118). The invention also providespolynucleotides and expression vectors encoding the mutantalpha-synuclein or homologue thereof, as well as cells comprising thepolynucleotide or expression vector. Finally, the invention provides anon-human animal comprising the mutant alpha-synuclein or homologuethereof. Such cells and non-human animals are particularly useful foridentifying substances that might prevent or reduce the toxicity ofalpha-synuclein.

BACKGROUND OF THE INVENTION

Parkinson's disease (PD) and several other neurodegenerative diseasesare degenerative disorders of the central nervous system and are bothchronic and progressive. The prevalence of PD in Europeans is 1.6% inpersons over 65 years of age. However, more than 10% of the patients arediagnosed before the age of 50. In 1990, an estimated 4 million peoplewere suffering from PD. It is characterized by muscle rigidity, tremor,a slowing of physical movement (bradykinesia) and, in extreme cases, aloss of physical movement (akinesia). The primary symptoms are theresults of decreased stimulation of the motor cortex by the basalganglia, normally caused by the insufficient formation and action ofdopamine, which is produced in the dopaminergic neurons of the brain.Secondary symptoms may include high level cognitive dysfunction andsubtle language problems. Typical other symptoms include disorders ofmood, behavior, thinking, and sensation (non-motor symptoms). Patients'individual symptoms may be quite dissimilar and progression of thedisease is also distinctly individual.

The symptoms of Parkinson's disease result from the loss of dopaminergiccells in the region of the substantia nigra pars compacta. These neuronsproject to the striatum and their loss leads to alterations in theactivity of the neural circuits within the basal ganglia that regulatemovement, in essence an inhibition of the direct pathway (facilitatingmovement) and excitation of the indirect pathway (inhibiting movement).The lack of dopamine results in increased inhibition of the ventralanterior nucleus of the thalamus, which sends excitatory projections tothe motor cortex, thus leading to hypokinesia.

The pathological hallmark feature of Parkinson's disease (PD) andseveral other neurodegenerative disorders is the deposition ofintracytoplasmic neuronal inclusions termed Lewy bodies. The majorcomponent of Lewy bodies are amyloid fibrils of the proteinalpha-synuclein (alpha-S). Protecting neurons from the toxicity ofalpha-synuclein is a promising strategy for treating these diseases.

Related diseases (sometimes called Parkinson-plus diseases) includedementia with Lewy bodies (DLB). While idiopathic Parkinson's diseasepatients also have Lewy bodies in their brain tissue, the distributionis denser and more widespread in DLB. Even so, the relationship betweenParkinson disease, Parkinson disease with dementia (PDD), and dementiawith Lewy bodies (DLB) might be most accurately conceptualized as aspectrum, with a discrete area of overlap between each of the threedisorders. The common involvement of alpha-synuclein in diseases such asPD, PDD, multiple system atrophy and the Lewy body variant ofAlzheimer's disease has led to a classification of these disease underthe term synucleinopathies.

Mutations of alpha-S associated with familial PD (A30P, A53T, E46K) havean increased aggregation propensity in vitro (US 2007/0213253), inagreement with aggregation of alpha-S into fibrillar Lewy bodies invivo. However, the role different aggregated alpha-S species play forneurotoxicity in vivo is unclear. Loss of dopaminergic terminals wasobserved in the presence of non-fibrillar alpha-S inclusions in one lineof A53T alpha-S transgenic mice (Masliah et al. Science 287, 1265-1269(2000)), raising the possibility that pre-fibrillar intermediates in thealpha-S aggregation process may be pathogenic (Lashuel & Lansbury Q RevBiophys 39, 167-201 (2006)), potentially by pore formation in cellmembranes. However, transgenic mice overexpressing A30P alpha-S failedto exhibit neurodegeneration (Lee et al. Proc Natl Acad Sci USA 99,8968-8973 (2002)), despite the fact that A30P alpha-S delays conversionof pre-fibrillar aggregates to fibrils (Conway et al., supra). Thus, theA30P alpha-S mice provided in vivo support that pre-fibrillar alpha-S isnot the primary toxic moiety.

US 2007/0192879 and WO 2008/063779 describe animal models and cellmodels overexpressing alpha-S. The only disclosed mutations are A30P,A53T and E46K, which are known to show an increased aggregationpropensity.

US 2007/0213253 disclose several synuclein mutants havingaggregation-inhibitory activity. Those mutants are supposed to becapable of inhibiting aggregation of wt alpha-S.

Also, Koo et al. Biochem. Biophys. Res. Comm. 368, 772-778 (2008)describe substitutions of alpha-synuclein which exhibit the capabilityto influence fibril formation of the protein.

Zhou et al. J. Biol. Chem. 283(15), 9863-9870 (2008) teaches tyrosine tocysteine substitutions in human alpha-synuclein showing enhancedalpha-synuclein fibril formation and neurotoxicity.

Thus, there is a need in the art for alpha-synuclein mutants that can beused in in vivo and in vitro screening assays in order to identifysubstances that can prevent or reduce the toxicity of alpha-synuclein,which plays a pivotal role in the pathology of synucleinopathies.

SUMMARY OF THE INVENTION

Accordingly, the present invention relates to a mutant humanalpha-synuclein with increased toxicity compared to wild-typealpha-synuclein, or a homologue thereof, wherein the mutantalpha-synuclein or homologue thereof comprises at least one amino acidsubstitution selected from the group consisting of a substitution at thealanine at position 56 (A56), at the alanine at position 76 (A76), atthe methionine at position 127 (M127) and/or at the valine at position118 (V118), as defined in the claims. Further, the invention relates toa polynucleotide encoding the mutant alpha-synuclein or homologuethereof, or an expression vector comprising said polynucleotide, a cellcomprising the polynucleotide or expression vector, as defined in theclaims. Also, a non-human animal comprising the cell of the invention isprovided, as defined in the claims. Finally, the invention providesmethods for identifying a substance that prevents or reduces toxicity ofalpha-synuclein, as defined in the claims.

DETAILED DESCRIPTION OF THE PREFERRED EMBODIMENTS

Little is known about the pathogenicity and the mechanism of toxicity ofaggregated alpha-S species, in particular in neuronal cells. To addressthese questions, the inventors generated alpha-S variants by astructure-based rational design and tested their biophysical andfunctional properties with respect to both fibril formation in vitro andtheir in vivo effect on neuronal activity and survival (toxicity) infour different model systems for PD, including both invertebrates(Caenorhabditis elegans, Drosophila melanogaster) and mammalian neurons.Surprisingly, it was found that the designed alpha-S variants of theinvention form soluble oligomers of defined sizes but cause indeed adramatic delay in fibril formation. Expression of pre-fibrillar alpha-Smutants in animal PD models affects locomotion, sleeping behaviour aswell as the lifespan of the animals, and causes neuronal toxicity.

The results shown herein provide insights into the role of multimericalpha-S species for PD pathogenesis and establish a tight link betweenthe biophysical properties of the alpha-S species and their function indifferent in vivo models. The newly established toxicity model andanimal models for alpha-S species provide a powerful tool for theidentification of potential therapeutic compounds.

Thus, in a first aspect, the present invention relates to a mutantalpha-synuclein with increased toxicity compared to wild-typealpha-synuclein having the amino acid sequence shown in SEQ ID NO: 1, ora homologue thereof, wherein the mutant alpha-synuclein or homologuethereof comprises at least one amino acid substitution selected from thegroup consisting of a substitution at the alanine at position 56 (A56),at the alanine at position 76 (A76), at the methionine at position 127(M127) and/or at the valine at position 118 (V118).

The present invention contemplates a mutant alpha-synuclein withdecreased fibril formation ability compared to wild-type alpha-synucleinhaving the amino acid sequence shown in SEQ ID NO: 1, or a homologuethereof, wherein the mutant alpha-synuclein or homologue thereofcomprises at least one amino acid substitution selected from the groupconsisting of a substitution at the alanine at position 56 (A56), at thealanine at position 76 (A76), at the methionine at position 127 (M127)and/or at the valine at position 118 (V118).

The present invention further contemplates a mutant alpha-synuclein withincreased toxicity and decreased fibril formation ability compared towild-type alpha-synuclein having the amino acid sequence shown in SEQ IDNO: 1, or a homologue thereof, wherein the mutant alpha-synuclein orhomologue thereof comprises at least one amino acid substitutionselected from the group consisting of a substitution at the alanine atposition 56 (A56), at the alanine at position 76 (A76), at themethionine at position 127 (M127) and/or at the valine at position 118(V118).

The present invention also contemplates a mutant alpha-synuclein withincreased toxicity compared to wild-type alpha-synuclein having theamino acid sequence shown in SEQ ID NO: 1, or a homologue thereof,wherein the mutant alpha-synuclein or homologue thereof comprises anamino acid substitution at the alanine at position 56 (A56) and/or atthe alanine at position 76 (A76).

Also, a mutant alpha-synuclein with decreased fibril formation abilitycompared to wild-type alpha-synuclein having the amino acid sequenceshown in SEQ ID NO: 1, or a homologue thereof, wherein the mutantalpha-synuclein or homologue thereof comprises an amino acidsubstitution at the alanine at position 56 (A56) and/or at the alanineat position 76 (A76) is contemplated.

In addition, a mutant alpha-synuclein with increased toxicity anddecreased fibril formation ability compared to wild-type alpha-synucleinhaving the amino acid sequence shown in SEQ ID NO: 1, or a homologuethereof, wherein the mutant alpha-synuclein or homologue thereofcomprises an amino acid substitution at the alanine at position 56 (A56)and/or at the alanine at position 76 (A76), is contemplated.

Thus, the mutant alpha-synuclein or homologue may comprise an amino acidsubstitution at the alanine at position 56 (or at an amino acid in thecorresponding position of the homologue) of SEQ ID NO: 1. The mutantalpha-synuclein or homologue of the first aspect may also comprise anamino acid substitution at the alanine at position 76 (or at an aminoacid in the corresponding position of the homologue) of SEQ ID NO: 1.The mutant alpha-synuclein or homologue of the first aspect may comprisean amino acid substitution at the alanine at position 56 (or at an aminoacid in the corresponding position of the homologue) and an amino acidsubstitution at the alanine at position 76 (or at an amino acid in thecorresponding position of the homologue) of SEQ ID NO: 1.

In one preferred embodiment of the present invention, the mutant orhomologue comprises an amino acid substitution at the alanine atposition 56 (A56) and at the alanine at position 76 (A76).

Also, the present invention contemplates a mutant alpha-synuclein withincreased toxicity compared to wild-type alpha-synuclein having theamino acid sequence shown in SEQ ID NO: 1, or a homologue thereof,wherein the mutant alpha-synuclein comprises an amino acid substitutionat the methionine at position 127 (M127) and/or the valine at position118 (V118), preferably wherein the methionine at position 127 and/or thevaline in the position 118 is substituted by alanine (A), glutamic acid(E), or aspartic acid (D), more preferably by alanine (A).

In addition, a mutant alpha-synuclein with increased toxicity anddecreased fibril formation ability compared to wild-type having theamino acid sequence shown in SEQ ID NO: 1, or a homologue thereof,wherein the mutant alpha-synuclein or homologue thereof comprises anamino acid substitution at the methionine at position 127 (M127) and/orthe valine at position 118 (V118), preferably wherein the methionine atposition 127 and/or the valine in the position 118 is substituted byalanine (A), glutamic acid (E), or aspartic acid (D), more preferably byalanine (A) is contemplated.

In one preferred embodiment of the present invention, the mutant orhomologue comprises an amino acid substitution at the methionine atposition 127 (M127) and at the valine at position 118 (V118).

It is also contemplated that these substitutions may be combined withany of the other embodiments described herein.

A mutant alpha-synuclein according to the invention relates to apolypeptide having at least 70% amino acid sequence identity on theamino acid level to SEQ ID NO: 1. Preferably, the mutant alpha-synucleinhas at least 71%, at least 72%, at least 73%, at least 74%, at least75%, at least 76%, at least 77%, at least 78%, at least 79%, at least80%, at least 81%, at least 82%, at least 83%, at least 84%, at least85%, at least 86%, at least 87%, at least 88%, at least 89%, at least90%, at least 91%, at least 92%, at least 93%, at least 94%, at least95%, at least 96%, at least 97%, at least 98%, at least 98.5%, at least99%, at least 99.5% amino acid sequence identity to SEQ ID NO: 1.

The term “mutant” alpha-synuclein implies that at least one single aminoacid residue of the alpha-synuclein molecule is deleted, inserted, orreplaced by another amino acid residue. The techniques of molecularbiology to generate these deletions, insertions or substitutions arewell-known in the art. Typically, the mutant alpha-synuclein does nothave 100% amino acid sequence identity to SEQ ID NO: 1. However, theremay be cases in which a program used for calculating sequence identityindicates 100% sequence identity between SEQ ID NO: 1 and anothersequence of interest, even though these two sequences differ by at leastone amino acid residue. In those cases, a mutant alpha-synuclein ispresent if there is a difference of at least one residue on the aminoacid sequence level between the sequence of interest and SEQ ID NO: 1.Optionally, the mutant alpha-synuclein may be a fusion proteincomprising a further protein component, such as a tag (e.g. forpurification), a marker (e.g. for localization), or an inoperable part(e.g. resulting from genetic manipulation) etc.

A polypeptide has “at least X % identity” to SEQ ID NO: 1 if SEQ ID NO:1 is aligned with the best matching sequence of a polypeptide ofinterest, and the amino acid identity between those two alignedsequences is at least X %. Such an alignment of amino acid sequences canbe performed using, for example, publicly available computer homologyprograms such as the “BLAST” program provided on the NCBI homepage atwww.ncbi.nlm.nih.gov/blast/blast.cgi, using the default settingsprovided therein. Further methods of calculating sequence identitypercentages of sets of amino acid sequences or nucleic acid sequencesare known in the art. The term “at position X”, always refers to theamino acid numbering according to SEQ ID NO: 1. An alignment of theamino acid sequence of a homologue with SEQ ID NO: 1 allows determininga corresponding amino acid (residue) in the homologue, e.g. thecorresponding amino acid residue(s) in the homologue to the amino acidresidue(s) which is/are substituted in SEQ ID NO: 1.

A “homologue” refers to an isoform of the alpha-synuclein of SEQ ID NO:1 or to an alpha-synuclein of a species other than human beingevolutionary homologous to the alpha-synuclein of SEQ ID NO: 1.Typically, the homologue has at least 70% amino acid sequence identityon the amino acid level to SEQ ID NO: 1. Preferably, the mutantalpha-synuclein has at least 71%, at least 72%, at least 73%, at least74%, at least 75%, at least 76%, at least 77%, at least 78%, at least79%, at least 80%, at least 81%, at least 82%, at least 83%, at least84%, at least 85%, at least 86%, at least 87%, at least 88%, at least89%, at least 90%, at least 91%, at least 92%, at least 93%, at least94%, at least 95%, at least 96%, at least 97%, at least 98%, at least98.5%, at least 99%, at least 99.5% or 100% (but still differing in atleast one amino acid residue, see above) amino acid sequence identity toSEQ ID NO: 1. More preferably, the mutant homologue of alpha-synucleinexhibits at least 30%, at least 35%, at least 40%, at least 45%, atleast 50%, at least 55%, at least 60%, at least 65%, at least 70%, atleast 75%, at least 80%, at least 85%, at least 86%, at least 87%, atleast 88%, at least 89%, at least 90%, at least 91%, at least 92%, atleast 93%, at least 94%, at least 95%, at least 96%, at least 97%, atleast 98%, at least 98.5%, at least 99%, at least 99.5% or 100% of thetoxicity of mutant alpha-synuclein of SEQ ID NO: 1, wherein the toxicitycan be determined as described below. Examples for homologues, which arenot intended to be limiting, are the alpha-synuclein of Pan troglodytes(accession number XP_(—)001162416.1), Pan paniscus (accession numberAAQ85068.1), Gorilla gorilla (accession number AAQ85072.1), Erythrocebuspatas (accession number AAQ85067.1), Macaca fascicularis (accessionnumber AAQ85071.1), Macaca mulatta (accession number AAQ85074), Pongoabelii (accession number AAQ85070.1), Saguinus labiatus (accessionnumber AAQ85075.1), Sus scrofa (accession number NP_(—)001032222.1),Lagothrix lagotricha (accession number AAQ85073.1), Ateles geoffroyi(accession number AAQ85076.1), Canis familiaris (accession numberXP_(—)535656.1), Rattus norvegicus (accession number NP_(—)062042.1),Mus musculus (accession number NP_(—)001035916), Bos Taurus (accessionnumber NP_(—) 001029213.1), Equus caballus (accession numberXP_(—)001496954.1), Gallus gallus (accession number NP_(—)990004.1),Taeniopygia guttata (accession number NP_(—) 001041718.1), Xenopuslaevis (accession number NP_(—) 001080623.1), and Xenopus tropicalis(accession number NP_(—)001090876.1).

In addition, the mutant alpha-synuclein or homologue has an increasedtoxicity compared to wild-type alpha-synuclein. The term “toxicity”describes the capability to which the mutant alpha-synuclein is able todamage a cell (cytotoxicity), an organ (organotoxicity) or a wholeorganism. In case the cell which is specifically damaged is a neuronalcell, in an isolated form or as a part of an organ or organism, thistype of toxicity is preferably called neurotoxicity. Thus, the term“toxicity” particularly encompasses the neurotoxicity of mutantalpha-synuclein. To test whether a mutant alpha-synuclein or homologuehas an increased toxicity compared to wild-type alpha-synuclein, invitro and in vivo assays can be performed.

For example, for an in vitro assay, a WST assay measuring mitochondrialdehydrogenase activity, can be performed according to the protocol ofthe manufacturer (Roche Diagnostics, Catalogue Number 1 644 807). Byusing a water-soluble tetrazolium salt (WST) colorimeteric assay formitochondrial dehydrogenase activity, toxicity of a mutantalpha-synuclein or homologue thereof to wt alpha-synuclein, eachtransfected into a neuronal cell, can be compared with a control cell.The WST colorimeteric assay estimates not only the mitochondrialcapacity to produce reduced equivalents but also the decline ofmitochondrial activity due to diminished cell numbers. Primary neuronalcells from rat embryos at E18 or E16 are transduced by AAV vectors atday 3 (DIV 3) and analysed at DIV 10. An increased toxicity is evidentif the cells transfected with the mutant-alpha-synuclein exhibit lessthan 90%, 85%, 80%, 75%, 70%, in particular less than 69%, 68%, 67%,66%, 65%, 64%, 63%, 62%, 61%, 60%, 59%, 58%, 57%, 56%, 55% mitochondrialdehydrogenase activity of the wild-type when compared under identicalconditions. Alternatively, toxicity may be determined in vitro by growthimpairment of yeast cells transfected with the mutant alpha-synuclein,wild-type alpha-synuclein and a vector control, as described in Example6.

Also, in vivo methods can be used for determining whether a mutantalpha-synuclein or homologue thereof has an increased toxicity comparedto wild-type. For example, transgenic C. elegans expressing wild-typealpha-synuclein or a mutant may be generated as described by Pitman, J.L., et al. Nature 441, 753-756 (2006). To image dopaminergic neurons,the animals are anesthetized by 50 mM sodium azide in M9 buffer andmounted on a 2% agarose pad. RFP positive dopaminergic neurons arevisualized using a Leica SP2 confocal microscope system. Neurite defectsare scored positive if one or more dendritic processes out of four havedegenerated. An increased toxicity is evident if less than 80%, 75%,70%, 65%, 60%, in particular 58%, 56%, 54%, 52%, 50%, 48%, 46%, 44%,42%, 40%, 38%, 36%, 34%, 32%, 30%, 28%, 26%, 24%, 22%, 20%, 18%, 16% ofthe worms expressing the mutant-alpha-synuclein are lacking neuritedefects when compared to the worms expressing wild-type alpha-synucleinunder identical conditions.

In another preferred embodiment, the mutant or homologue of theinvention may further comprise the substitution A30P.

The alanine at position 56 and/or the alanine at position 76 may besubstituted by any amino acid other than alanine, or, in case of ahomologue, any amino acid other than the corresponding amino acid in thehomologue. Preferably the substitution is a non-conservativesubstitution, in particular wherein the alanine at position 56 and/orthe alanine at position 76 is substituted by glutamic acid (E), asparticacid (D), or a proline (P) residue, preferably by a proline residue.

A “conservative” amino acid exchange encompasses a conservativesubstitution of one residue by one of a certain set of other residues,such as indicated in the table below. Thus, if an alanine at theposition 56 or 76 of an alpha-synuclein molecule of the invention issubstituted by a non-conservative amino acid, Ala is substituted by anamino acid other than Gly, Ser, or Thr. Preferably, the alanine issubstituted by Glu (E), Asp (D), or Pro (P), and even more preferably,the alanine is substituted by Pro (P). Thus preferred substitutions areA56D, A56E, A56P, A76D, A76E, A76P, A56D+A76D, A56D+A76E, A56D+A76P,A56E+A76D, A56E+A76E, A56E+A76P, A56P+A76E, A56P+A76D, A56P+A76P, morepreferred substitutions are A56D+A76P, A56E+A76P, A56P+A76E, A56P+A76D,A56P+A76P, and most preferred substitutions are A56P, A76P, andA56P+A76P.

Amino acid Conservative substitution A G; S; T C A; V; L D E; N; Q E D;Q; N F W; Y; L; M; H G A H Y; F; K; R I V; L; M; A K R; H L M; I; V; A ML; I; V; A N Q P V; I Q N R K; H S A; T; G; N T A; S; G; N; V V A; L; IW F; Y; H Y F; W; H

In still another preferred embodiment, the substitution is anon-conservative substitution or a substitution by alanine, inparticular wherein the methionine at position 127 and/or the valine inthe position 118 is substituted by alanine (A), glutamic acid (E), oraspartic acid (D), more preferably by alanine (A). This means that anyof the above mentioned substitutions or combination of substitutions,either comprising the substitution A30P or not, may further comprise asubstitution at position M127 and/or V118, i.e. a substitution atposition M127, or a substitution at position V118, or a substitution atposition M127 and V118 of SEQ ID NO: 1, or at the corresponding aminoacid position(s) of the homologue. M127 and/or V118 may be substitutedby any amino acid other than methionine or valine, respectively, or anyamino acid other than the corresponding amino acid in the homologue.However, preferred substitutions are M127A, M127E, M127D, V118A, V118E,V188D, M127A+V118A, M127A+V118E, M127A+V188D, M127E+V118A, M127E+V118E,M127E+V118D, M127D+V118A, M127D+V118E, M127D+V118D, even more preferredsubstitutions are M127A, V118A, M127A+V118A, M127A+V118E, M127A+V118D,M127E+V118A, M127D+V118A and most preferred substitutions are M127A,V118A, M127A+V118A.

Further preferred substitutions are:

A56D+M127A, A56E+M127A, A56P+M127A, A76D+M127A, A76E+M127A, A76P+M127A,A56D+A76D+M127A, A56D+A76E+M127A, A56D+A76P+M127A, A56E+A76D+M127A,A56E+A76E+M127A, A56E+A76P+M127A, A56P+A76E+M127A, A56P+A76D+M127A,A56P+A76P+M127A, A56D+A76P+M127A, A56E+A76P+M127A, A56P+A76E+M127A,A56P+A76D+M127A, A56P+A76P+M127A, A56P+M127A, A76P+M127A,A56P+A76P+M127A,

A56D+M127E, A56E+M127E, A56P+M127E, A76D+M127E, A76E+M127E, A76P+M127E,A56D+A76D+M127E, A56D+A76E+M127E, A56D+A76P+M127E, A56E+A76D+M127E,A56E+A76E+M127E, A56E+A76P+M127E, A56P+A76E+M127E, A56P+A76D+M127E,A56P+A76P+M127E, A56D+A76P+M127E, A56E+A76P+M127E, A56P+A76E+M127E,A56P+A76D+M127E, A56P+A76P+M127E, A56P+M127E, A76P+M127E,A56P+A76P+M127E,

A56D+M127D, A56E+M127D, A56P+M127D, A76D+M127D, A76E+M127D, A76P+M127D,A56D+A76D+M127D, A56D+A76E+M127D, A56D+A76P+M127D, A56E+A76D+M127D,A56E+A76E+M127D, A56E+A76P+M127D, A56P+A76E+M127D, A56P+A76D+M127D,A56P+A76P+M127D, A56D+A76P+M127D, A56E+A76P+M127D, A56P+A76E+M127D,A56P+A76D+M127D, A56P+A76P+M127D, A56P+M127D, A76P+M127D,A56P+A76P+M127D,

A56D+V118A, A56E+V118A, A56P+V118A, A76D+V118A, A76E+V118A, A76P+V118A,A56D+A76D+V118A, A56D+A76E+V118A, A56D+A76P+V118A, A56E+A76D+V118A,A56E+A76E+V118A, A56E+A76P+V118A, A56P+A76E+V118A, A56P+A76D+V118A,A56P+A76P+V118A, A56D+A76P+V118A, A56E+A76P+V118A, A56P+A76E+V118A,A56P+A76D+V118A, A56P+A76P+V118A, A56P+V118A, A76P+V118A,A56P+A76P+V118A,

A56D+V118E, A56E+V118E, A56P+V118E, A76D+V118E, A76E+V118E, A76P+V118E,A56D+A76D+V118E, A56D+A76E+V118E, A56D+A76P+V118E, A56E+A76D+V118E,A56E+A76E+V118E, A56E+A76P+V118E, A56P+A76E+V118E, A56P+A76D+V118E,A56P+A76P+V118E, A56D+A76P+V118E, A56E+A76P+V118E, A56P+A76E+V118E,A56P+A76D+V118E, A56P+A76P+V118E, A56P+V118E, A76P+V118E,A56P+A76P+V118E,

A56D+V118D, A56E+V118D, A56P+V118D, A76D+V118D, A76E+V118D, A76P+V118D,A56D+A76D+V118D, A56D+A76E+V118D, A56D+A76P+V118D, A56E+A76D+V118D,A56E+A76E+V118D, A56E+A76P+V118D, A56P+A76E+V118D, A56P+A76D+V118D,A56P+A76P+V118D, A56D+A76P+V118D, A56E+A76P+V118D, A56P+A76E+V118D,A56P+A76D+V118D, A56P+A76P+V118D, A56P+V118D, A76P+V118D,A56P+A76P+V118D,

A56D+M127A+V118A, A56E+M127A+V118A, A56P+M127A+V118A, A76D+M127A+V118A,A76E+M127A+V118A, A76P+M127A+V118A, A56D+A76D+M127A+V118A,A56D+A76E+M127A+V118A, A56D+A76P+M127A+V118A, A56E+A76D+M127A+V118A,A56E+A76E+M127A+V118A, A56E+A76P+M127A+V118A, A56P+A76E+M127A+V118A,A56P+A76D+M127A+V118A, A56P+A76P+M127A+V118A, A56D+A76P+M127A+V118A,A56E+A76P+M127A+V118A, A56P+A76E+M127A+V118A, A56P+A76D+M127A+V118A,A56P+A76P+M127A+V118A, A56P+M127A+V118A, A76P+M127A+V118A,A56P+A76P+M127A+V118A,

A56D+M127A+V118E, A56E+M127A+V118E, A56P+M127A+V118E, A76D+M127A+V118E,A76E+M127A+V118E, A76P+M127A+V118E, A56D+A76D+M127A+V118E,A56D+A76E+M127A+V118E, A56D+A76P+M127A+V118E, A56E+A76D+M127A+V118E,A56E+A76E+M127A+V118E, A56E+A76P+M127A+V118E, A56P+A76E+M127A+V118E,A56P+A76D+M127A+V118E, A56P+A76P+M127A+V118E, A56D+A76P+M127A+V118E,A56E+A76P+M127A+V118E, A56P+A76E+M127A+V118E, A56P+A76D+M127A+V118E,A56P+A76P+M127A+V118E, A56P+M127A+V118E, A76P+M127A+V118E,A56P+A76P+M127A+V118E,

A56D+M127A+V118D, A56E+M127A+V118D, A56P+M127A+V118D, A76D+M127A+V118D,A76E+M127A+V118D, A76P+M127A+V118D, A56D+A76D+M127A+V118D,A56D+A76E+M127A+V118D, A56D+A76P+M127A+V118D, A56E+A76D+M127A+V118D,A56E+A76E+M127A+V118D, A56E+A76P+M127A+V118D, A56P+A76E+M127A+V118D,A56P+A76D+M127A+V118D, A56P+A76P+M127A+V118D, A56D+A76P+M127A+V118D,A56E+A76P+M127A+V118D, A56P+A76E+M127A+V118D, A56P+A76D+M127A+V118D,A56P+A76P+M127A+V118D, A56P+M127A+V118D, A76P+M127A+V118D,A56P+A76P+M127A+V118D,

A56D+M127E+V118A, A56E+M127E+V118A, A56P+M127E+V118A, A76D+M127E+V118A,A76E+M127E+V118A, A76P+M127E+V118A, A56D+A76D+M127E+V118A,A56D+A76E+M127E+V118A, A56D+A76P+M127E+V118A, A56E+A76D+M127E+V118A,A56E+A76E+M127E+V118A, A56E+A76P+M127E+V118A, A56P+A76E+M127E+V118A,A56P+A76D+M127E+V118A, A56P+A76P+M127E+V118A, A56D+A76P+M127E+V118A,A56E+A76P+M127E+V118A, A56P+A76E+M127E+V118A, A56P+A76D+M127E+V118A,A56P+A76P+M127E+V118A, A56P+M127E+V118A, A76P+M127E+V118A,A56P+A76P+M127E+V118A,

A56D+M127E+V118E, A56E+M127E+V118E, A56P+M127E+V118E, A76D+M127E+V118E,A76E+M127E+V118E, A76P+M127E+V118E, A56D+A76D+M127E+V118E,A56D+A76E+M127E+V118E, A56D+A76P+M127E+V118E, A56E+A76D+M127E+V118E,A56E+A76E+M127E+V118E, A56E+A76P+M127E+V118E, A56P+A76E+M127E+V118E,A56P+A76D+M127E+V118E, A56P+A76P+M127E+V118E, A56D+A76P+M127E+V118E,A56E+A76P+M127E+V118E, A56P+A76E+M127E+V118E, A56P+A76D+M127E+V118E,A56P+A76P+M127E+V118E, A56P+M127E+V118E, A76P+M127E+V118E,A56P+A76P+M127E+V118E,

A56D+M127E+V118D, A56E+M127E+V118D, A56P+M127E+V118D, A76D+M127E+V118D,A76E+M127E+V118D, A76P+M127E+V118D, A56D+A76D+M127E+V118D,A56D+A76E+M127E+V118D, A56D+A76P+M127E+V118D, A56E+A76D+M127E+V118D,A56E+A76E+M127E+V118D, A56E+A76P+M127E+V118D, A56P+A76E+M127E+V118D,A56P+A76D+M127E+V118D, A56P+A76P+M127E+V118D, A56D+A76P+M127E+V118D,A56E+A76P+M127E+V118D, A56P+A76E+M127E+V118D, A56P+A76D+M127E+V118D,A56P+A76P+M127E+V118D, A56P+M127E+V118D, A76P+M127E+V118D,A56P+A76P+M127E+V118D,

A56D+M127D+V118A, A56E+M127D+V118A, A56P+M127D+V118A, A76D+M127D+V118A,A76E+M127D+V118A, A76P+M127D+V118A, A56D+A76D+M127D+V118A,A56D+A76E+M127D+V118A, A56D+A76P+M127D+V118A, A56E+A76D+M127D+V118A,A56E+A76E+M127D+V118A, A56E+A76P+M127D+V118A, A56P+A76E+M127D+V118A,A56P+A76D+M127D+V118A, A56P+A76P+M127D+V118A, A56D+A76P+M127D+V118A,A56E+A76P+M127D+V118A, A56P+A76E+M127D+V118A, A56P+A76D+M127D+V118A,A56P+A76P+M127D+V118A, A56P+M127D+V118A, A76P+M127D+V118A,A56P+A76P+M127D+V118A,

A56D+M127D+V118E, A56E+M127D+V118E, A56P+M127D+V118E, A76D+M127D+V118E,A76E+M127D+V118E, A76P+M127D+V118E, A56D+A76D+M127D+V118E,A56D+A76E+M127D+V118E, A56D+A76P+M127D+V118E, A56E+A76D+M127D+V118E,A56E+A76E+M127D+V118E, A56E+A76P+M127D+V118E, A56P+A76E+M127D+V118E,A56P+A76D+M127D+V118E, A56P+A76P+M127D+V118E, A56D+A76P+M127D+V118E,A56E+A76P+M127D+V118E, A56P+A76E+M127D+V118E, A56P+A76D+M127D+V118E,A56P+A76P+M127D+V118E, A56P+M127D+V118E, A76P+M127D+V118E,A56P+A76P+M127D+V118E,

A56D+M127D+V118D, A56E+M127D+V118D, A56P+M127D+V118D, A76D+M127D+V118D,A76E+M127D+V118D, A76P+M127D+V118D, A56D+A76D+M127D+V118D,A56D+A76E+M127D+V118D, A56D+A76P+M127D+V118D, A56E+A76D+M127D+V118D,A56E+A76E+M127D+V118D, A56E+A76P+M127D+V118D, A56P+A76E+M127D+V118D,A56P+A76D+M127D+V118D, A56P+A76P+M127D+V118D, A56D+A76P+M127D+V118D,A56E+A76P+M127D+V118D, A56P+A76E+M127D+V118D, A56P+A76D+M127D+V118D,A56P+A76P+M127D+V118D, A56P+M127D+V118D, A76P+M127D+V118D,A56P+A76P+M127D+V118D,

A56D+M127A+A30P, A56E+M127A+A30P, A56P+M127A+A30P, A76D+M127A+A30P,A76E+M127A+A30P, A76P+M127A+A30P, A56D+A76D+M127A+A30P,A56D+A76E+M127A+A30P, A56D+A76P+M127A+A30P, A56E+A76D+M127A+A30P,A56E+A76E+M127A+A30P, A56E+A76P+M127A+A30P, A56P+A76E+M127A+A30P,A56P+A76D+M127A+A30P, A56P+A76P+M127A+A30P, A56D+A76P+M127A+A30P,A56E+A76P+M127A+A30P, A56P+A76E+M127A+A30P, A56P+A76D+M127A+A30P,A56P+A76P+M127A+A30P, A56P+M127A+A30P, A76P+M127A+A30P,A56P+A76P+M127A+A30P,

A56D+M127E+A30P, A56E+M127E+A30P, A56P+M127E+A30P, A76D+M127E+A30P,A76E+M127E+A30P, A76P+M127E+A30P, A56D+A76D+M127E+A30P,A56D+A76E+M127E+A30P, A56D+A76P+M127E+A30P, A56E+A76D+M127E+A30P,A56E+A76E+M127E+A30P, A56E+A76P+M127E+A30P, A56P+A76E+M127E+A30P,A56P+A76D+M127E+A30P, A56P+A76P+M127E+A30P, A56D+A76P+M127E+A30P,A56E+A76P+M127E+A30P, A56P+A76E+M127E+A30P, A56P+A76D+M127E+A30P,A56P+A76P+M127E+A30P, A56P+M127E+A30P, A76P+M127E+A30P,A56P+A76P+M127E+A30P,

A56D+M127D+A30P, A56E+M127D+A30P, A56P+M127D+A30P, A76D+M127D+A30P,A76E+M127D+A30P, A76P+M127D+A30P, A56D+A76D+M127D+A30P,A56D+A76E+M127D+A30P, A56D+A76P+M127D+A30P, A56E+A76D+M127D+A30P,A56E+A76E+M127D+A30P, A56E+A76P+M127D+A30P, A56P+A76E+M127D+A30P,A56P+A76D+M127D+A30P, A56P+A76P+M127D+A30P, A56D+A76P+M127D+A30P,A56E+A76P+M127D+A30P, A56P+A76E+M127D+A30P, A56P+A76D+M127D+A30P,A56P+A76P+M127D+A30P, A56P+M127D+A30P, A76P+M127D+A30P,A56P+A76P+M127D+A30P,

A56D+V118A+A30P, A56E+V118A+A30P, A56P+V118A+A30P, A76D+V118A+A30P,A76E+V118A+A30P, A76P+V118A+A30P, A56D+A76D+V118A+A30P,A56D+A76E+V118A+A30P, A56D+A76P+V118A+A30P, A56E+A76D+V118A+A30P,A56E+A76E+V118A+A30P, A56E+A76P+V118A+A30P, A56P+A76E+V118A+A30P,A56P+A76D+V118A+A30P, A56P+A76P+V118A+A30P, A56D+A76P+V118A+A30P,A56E+A76P+V118A+A30P, A56P+A76E+V118A+A30P, A56P+A76D+V118A+A30P,A56P+A76P+V118A+A30P, A56P+V118A+A30P, A76P+V118A+A30P,A56P+A76P+V118A+A30P,

A56D+V118E+A30P, A56E+V118E+A30P, A56P+V118E+A30P, A76D+V118E+A30P,A76E+V118E+A30P, A76P+V118E+A30P, A56D+A76D+V118E+A30P,A56D+A76E+V118E+A30P, A56D+A76P+V118E+A30P, A56E+A76D+V118E+A30P,A56E+A76E+V118E+A30P, A56E+A76P+V118E+A30P, A56P+A76E+V118E+A30P,A56P+A76D+V118E+A30P, A56P+A76P+V118E+A30P, A56D+A76P+V118E+A30P,A56E+A76P+V118E+A30P, A56P+A76E+V118E+A30P, A56P+A76D+V118E+A30P,A56P+A76P+V118E+A30P, A56P+V118E+A30P, A76P+V118E+A30P,A56P+A76P+V118E+A30P,

A56D+V118D+A30P, A56E+V118D+A30P, A56P+V118D+A30P, A76D+V118D+A30P,A76E+V118D+A30P, A76P+V118D+A30P, A56D+A76D+V118D+A30P,A56D+A76E+V118D+A30P, A56D+A76P+V118D+A30P, A56E+A76D+V118D+A30P,A56E+A76E+V118D+A30P, A56E+A76P+V118D+A30P, A56P+A76E+V118D+A30P,A56P+A76D+V118D+A30P, A56P+A76P+V118D+A30P, A56D+A76P+V118D+A30P,A56E+A76P+V118D+A30P, A56P+A76E+V118D+A30P, A56P+A76D+V118D+A30P,A56P+A76P+V118D+A30P, A56P+V118D+A30P, A76P+V118D+A30P,A56P+A76P+V118D+A30P,

A56D+M127A+V118A+A30P, A56E+M127A+V118A+A30P, A56P+M127A+V118A+A30P,A76D+M127A+V118A+A30P, A76E+M127A+V118A+A30P, A76P+M127A+V118A+A30P,A56D+A76D+M127A+V118A+A30P, A56D+A76E+M127A+V118A+A30P,A56D+A76P+M127A+V118A+A30P, A56E+A76D+M127A+V118A+A30P,A56E+A76E+M127A+V118A+A30P, A56E+A76P+M127A+V118A+A30P,A56P+A76E+M127A+V118A+A30P, A56P+A76D+M127A+V118A+A30P,A56P+A76P+M127A+V118A+A30P, A56D+A76P+M127A+V118A+A30P,A56E+A76P+M127A+V118A+A30P, A56P+A76E+M127A+V118A+A30P,A56P+A76D+M127A+V118A+A30P, A56P+A76P+M127A+V118A+A30P,A56P+M127A+V118A+A30P, A76P+M127A+V118A+A30P,A56P+A76P+M127A+V118A+A30P,

A56D+M127A+V118E+A30P, A56E+M127A+V118E+A30P, A56P+M127A+V118E+A30P,A76D+M127A+V118E+A30P, A76E+M127A+V118E+A30P, A76P+M127A+V118E+A30P,A56D+A76D+M127A+V118E+A30P, A56D+A76E+M127A+V118E+A30P,A56D+A76P+M127A+V118E+A30P, A56E+A76D+M127A+V118E+A30P,A56E+A76E+M127A+V118E+A30P, A56E+A76P+M127A+V118E+A30P,A56P+A76E+M127A+V118E+A30P, A56P+A76D+M127A+V118E+A30P,A56P+A76P+M127A+V118E+A30P, A56D+A76P+M127A+V118E+A30P,A56E+A76P+M127A+V118E+A30P, A56P+A76E+M127A+V118E+A30P,A56P+A76D+M127A+V118E+A30P, A56P+A76P+M127A+V118E+A30P,A56P+M127A+V118E+A30P, A76P+M127A+V118E+A30P,A56P+A76P+M127A+V118E+A30P,

A56D+M127A+V118D+A30P, A56E+M127A+V118D+A30P, A56P+M127A+V118D+A30P,A76D+M127A+V118D+A30P, A76E+M127A+V118D+A30P, A76P+M127A+V118D+A30P,A56D+A76D+M127A+V118D+A30P, A56D+A76E+M127A+V118D+A30P,A56D+A76P+M127A+V118D+A30P, A56E+A76D+M127A+V118D+A30P,A56E+A76E+M127A+V118D+A30P, A56E+A76P+M127A+V118D+A30P,A56P+A76E+M127A+V118D+A30P, A56P+A76D+M127A+V118D+A30P,A56P+A76P+M127A+V118D+A30P, A56D+A76P+M127A+V118D+A30P,A56E+A76P+M127A+V118D+A30P, A56P+A76E+M127A+V118D+A30P,A56P+A76D+M127A+V118D+A30P, A56P+A76P+M127A+V118D+A30P,A56P+M127A+V118D+A30P, A76P+M127A+V118D+A30P,A56P+A76P+M127A+V118D+A30P,

A56D+M127E+V118A+A30P, A56E+M127E+V118A+A30P, A56P+M127E+V118A+A30P,A76D+M127E+V118A+A30P, A76E+M127E+V118A+A30P, A76P+M127E+V118A+A30P,A56D+A76D+M127E+V118A+A30P, A56D+A76E+M127E+V118A+A30P,A56D+A76P+M127E+V118A+A30P, A56E+A76D+M127E+V118A+A30P,A56E+A76E+M127E+V118A+A30P, A56E+A76P+M127E+V118A+A30P,A56P+A76E+M127E+V118A+A30P, A56P+A76D+M127E+V118A+A30P,A56P+A76P+M127E+V118A+A30P, A56D+A76P+M127E+V118A+A30P,A56E+A76P+M127E+V118A+A30P, A56P+A76E+M127E+V118A+A30P,A56P+A76D+M127E+V118A+A30P, A56P+A76P+M127E+V118A+A30P,A56P+M127E+V118A+A30P, A76P+M127E+V118A+A30P,A56P+A76P+M127E+V118A+A30P,

A56D+M127E+V118E+A30P, A56E+M127E+V118E+A30P, A56P+M127E+V118E+A30P,A76D+M127E+V118E+A30P, A76E+M127E+V118E+A30P, A76P+M127E+V118E+A30P,A56D+A76D+M127E+V118E+A30P, A56D+A76E+M127E+V118E+A30P,A56D+A76P+M127E+V118E+A30P, A56E+A76D+M127E+V118E+A30P,A56E+A76E+M127E+V118E+A30P, A56E+A76P+M127E+V118E+A30P,A56P+A76E+M127E+V118E+A30P, A56P+A76D+M127E+V118E+A30P,A56P+A76P+M127E+V118E+A30P, A56D+A76P+M127E+V118E+A30P,A56E+A76P+M127E+V118E+A30P, A56P+A76E+M127E+V118E+A30P,A56P+A76D+M127E+V118E+A30P, A56P+A76P+M127E+V118E+A30P,A56P+M127E+V118E+A30P, A76P+M127E+V118E+A30P,A56P+A76P+M127E+V118E+A30P,

A56D+M127E+V118D+A30P, A56E+M127E+V118D+A30P, A56P+M127E+V118D+A30P,A76D+M127E+V118D+A30P, A76E+M127E+V118D+A30P, A76P+M127E+V118D+A30P,A56D+A76D+M127E+V118D+A30P, A56D+A76E+M127E+V118D+A30P,A56D+A76P+M127E+V118D+A30P, A56E+A76D+M127E+V118D+A30P,A56E+A76E+M127E+V118D+A30P, A56E+A76P+M127E+V118D+A30P,A56P+A76E+M127E+V118D+A30P, A56P+A76D+M127E+V118D+A30P,A56P+A76P+M127E+V118D+A30P, A56D+A76P+M127E+V118D+A30P,A56E+A76P+M127E+V118D+A30P, A56P+A76E+M127E+V118D+A30P,A56P+A76D+M127E+V118D+A30P, A56P+A76P+M127E+V118D+A30P,A56P+M127E+V118D+A30P, A76P+M127E+V118D+A30P,A56P+A76P+M127E+V118D+A30P,

A56D+M127D+V118A+A30P, A56E+M127D+V118A+A30P, A56P+M127D+V118A+A30P,A76D+M127D+V118A+A30P, A76E+M127D+V118A+A30P, A76P+M127D+V118A+A30P,A56D+A76D+M127D+V118A+A30P, A56D+A76E+M127D+V118A+A30P,A56D+A76P+M127D+V118A+A30P, A56E+A76D+M127D+V118A+A30P,A56E+A76E+M127D+V118A+A30P, A56E+A76P+M127D+V118A+A30P,A56P+A76E+M127D+V118A+A30P, A56P+A76D+M127D+V118A+A30P,A56P+A76P+M127D+V118A+A30P, A56D+A76P+M127D+V118A+A30P,A56E+A76P+M127D+V118A+A30P, A56P+A76E+M127D+V118A+A30P,A56P+A76D+M127D+V118A+A30P, A56P+A76P+M127D+V118A+A30P,A56P+M127D+V118A+A30P, A76P+M127D+V118A+A30P,A56P+A76P+M127D+V118A+A30P,

A56D+M127D+V118E+A30P, A56E+M127D+V118E+A30P, A56P+M127D+V118E+A30P,A76D+M127D+V118E+A30P, A76E+M127D+V118E+A30P, A76P+M127D+V118E+A30P,A56D+A76D+M127D+V118E+A30P, A56D+A76E+M127D+V118E+A30P,A56D+A76P+M127D+V118E+A30P, A56E+A76D+M127D+V118E+A30P,A56E+A76E+M127D+V118E+A30P, A56E+A76P+M127D+V118E+A30P,A56P+A76E+M127D+V118E+A30P, A56P+A76D+M127D+V118E+A30P,A56P+A76P+M127D+V118E+A30P, A56D+A76P+M127D+V118E+A30P,A56E+A76P+M127D+V118E+A30P, A56P+A76E+M127D+V118E+A30P,A56P+A76D+M127D+V118E+A30P, A56P+A76P+M127D+V118E+A30P,A56P+M127D+V118E+A30P, A76P+M127D+V118E+A30P,A56P+A76P+M127D+V118E+A30P,

A56D+M127D+V118D+A30P, A56E+M127D+V118D+A30P, A56P+M127D+V118D+A30P,A76D+M127D+V118D+A30P, A76E+M127D+V118D+A30P, A76P+M127D+V118D+A30P,A56D+A76D+M127D+V118D+A30P, A56D+A76E+M127D+V118D+A30P,A56D+A76P+M127D+V118D+A30P, A56E+A76D+M127D+V118D+A30P,A56E+A76E+M127D+V118D+A30P, A56E+A76P+M127D+V118D+A30P,A56P+A76E+M127D+V118D+A30P, A56P+A76D+M127D+V118D+A30P,A56P+A76P+M127D+V118D+A30P, A56D+A76P+M127D+V118D+A30P,A56E+A76P+M127D+V118D+A30P, A56P+A76E+M127D+V118D+A30P,A56P+A76D+M127D+V118D+A30P, A56P+A76P+M127D+V118D+A30P,A56P+M127D+V118D+A30P, A76P+M127D+V118D+A30P,A56P+A76P+M127D+V118D+A30P,

In addition to the mutation A30P in human alpha synuclein, also A53T andE46K are believed to be involved in the development of Parkinson'sdisease (US 2007/0213253).

Koo et al. Biochem. Biophys. Res. Comm. 368, 772-778 (2008) showsbiophysical data substitutions of alpha-synuclein which exhibit thecapability to influence fibril formation of the protein. In particularsubstitutions V37P, L38P, V40P, V48P, V49P, V52P, T59P, V63P, T64P,N65P, G67P, G68P, A69P, V70P, T72P, T75P, T81P, E83P, A89P, A90P showeddelayed aggregation of the alpha-synuclein protein. Furthermoresubstitutions T72E, T75K, V95S and A78T have been found to delay fibrilformation in vitro.

A human alpha-synuclein having a Y39C substitution was recently shown byZhou et al. J. Biol. Chem. 283(15), 9863-9870 (2008) to enhancealpha-synuclein fibril formation and neurotoxicity.

US 2007/0213253 discloses substitutions in the human alpha-synucleinresulting in mutants with decreased ability of forming aggregation. Inparticular, the substitutions A50P, Gly68T, Gly68V, A69T, A69V, A69K,V70T, V70P, V70F, V71T, V71K, T72V, T72E, V74T, V77T and V82K aredisclosed.

Moreover, further point mutations which influence fibrillation ofalpha-synuclein are known: E13K, Q24K, E35K, Y39A, K45E, V40D, E61K,V66S, V66P, V70G, V74D, V74E, V74G, E83K, Y125A, Y133A, Y136A (Harada etal. Biochim. Biophys. Acta, Epub July 2009; Kumar et al. Biochem.Biophys. Res. Commun., Epub July 2009; Koo et al. Biochem. Biophys. Res.Commun. 386(1): 165-169 (2009); Ulrih et al. Biochim. Biophys. Acta1782(10): 581-585 (2008)).

However, it is considered highly likely that also other substitutions(e.g. of amino acid positions 53-55, 57-89, 73-75, 77-80) or theintroduction of charged residues in the central domain ofalpha-synuclein (residues 30 to 100), which forms the core of thefibrils, may have a negative effect on the fibril formation and/or anincreased toxicity.

Accordingly, in a further preferred embodiment, the mutant or homologueof the invention may further comprise at least one of the substitutionsE13K, Q24K, E35K, V37P, L38P, Y39C, Y39A, V40P, V40D, K45E, E46K, V48P,V49P, A50P, V52P, A53T, T59P, E61K, V63P, T64P, N65P, V66S, V66P, G67P,G68P, G68T, G68V, A69P, A69T, A69V, A69K, V70G, V70T, V70P, V70F, V71T,V71K, T72P, T72V, T72E, V74D, V74E, V74G, V74T, T75P, T75K, V77T, A78T,T81P, V82K, E83K, E83P, A89P, A90P, V95S, Y125A, Y133A, Y136A.Consequently, this means that the mutant or homologue comprising atleast one amino acid substitution selected from the group consisting ofa substitution at the alanine at position 56 (A56), at the alanine atposition 76 (A76), at the methionine at position 127 (M127) and/or atthe valine at position 118 (V118), either further comprising thesubstitution A30P or not, may further comprise at least one of thesesubstitutions in SEQ ID NO: 1 or at the corresponding amino acidresidue(s) of the homologue.

“At least one substitution” means that the mutant alpha-synuclein maycomprise one, or two, or three, or four, or five, or six, or seven, oreight, or nine, or ten, or eleven, or twelve, or 13, or 14, or 15, oreven more substitutions selected from the group of substitutions givenabove.

In another preferred embodiment, the mutant or homologue according tothe invention may be one, wherein 1 to 40 amino acids of the C-terminalend are deleted. Thus, 1 to 40 amino acids of the residues 100 to 140 ofSEQ ID NO: 1 may be deleted, preferably a stretch of 1 to 40 aminoacids, including residue 140 of SEQ ID NO: 1. However, the 1 to 40 aminoacids to be deleted may also not be in one stretch and may also notcontain residue 140 of SEQ ID NO: 1. Preferably up to 1, 2, 3, 4, 5, 6,7, 8, 9, 10, 11, 12, 13, 14, 15, 20, 21, 25, 30, or 35 amino acids ofthe C-terminal end are deleted.

In one preferred embodiment, the mutant or homologue of the inventionmay be one, wherein the fibril formation ability of the mutantalpha-synuclein or homologue is decreased compared to wild-typealpha-synuclein. If the fibril formation ability is decreased comparedto the wild-type can be determined using the Thioflavin (ThioT)fluorescence assay. Briefly, 5 μl aliquots (100 μM) from mutant andwild-type alpha-synuclein incubations are withdrawn and added to 2 ml of5 μM ThioT in 50 mM Glycine-NaOH pH 8.2. Fluorescence measurements arecarried out on a spectrofluorometer using 3.5 ml quartz cuvettes with apath length of 1 cm. Fluorescence emission spectra are recorded from 465to 600 nm, using excitation wavelength of 446 nm, an integration time of0.1 second, and both excitation and emission bandwidths of 10 nm.Kinetic aggregation traces are generated from time traces of ThioTfluorescence intensity at 482 nm and corrected for free ThioTfluorescence. Aggregation yields are normalized to the final values andthe averaged data points are fitted to a sigmoidal equation. A decreasedfibril formation ability is evident if the lag time is at least 2fold,5fold, 10fold, 20fold, 50fold, 60fold, 70fold, 80fold, 90fold, 100fold,more preferably 200fold, 300fold, 400fold, 500fold, 600fold, 800fold,900fold, 1000fold, 10000fold, 100000fold higher compared to wild-typealpha-synuclein under identical conditions. Most preferably, the mutantalpha-synuclein or homologue has no more fibril formation ability, eventhough pre-fibrillar alpha-synuclein complexes may be formed.

In a further aspect, the invention relates to a polynucleotide encodingthe mutant alpha-synuclein or homologue thereof according to theinvention, or an expression vector comprising said polynucleotide.

A polynucleotide can be any nucleic acid sequence capable of encoding amutant alpha-synuclein or homologue according to the invention, such assingle-stranded or double-stranded DNA, the sense or antisense strand ofa DNA molecule, or RNA molecules, and the like. The person skilled inthe art knows how to derive a polynucleotide sequence coding for aprotein and how to isolate or produce such a nucleic acid sequence usingstandard techniques of molecular biology. Since the polynucleotideencoding the mutant alpha-synuclein or homologue does not encode anaturally occurring polypeptide (e.g., it encodes a mutated and/ortruncated alpha-synuclein or a fusion protein) the respective nucleicacid coding for the polypeptide may be produced in accordance withstandard procedures including well-known methods of genetic engineering.Further, the polynucleotide sequence may also be adapted to the codonusage of the host intended to be transfected with the polynucleotide.

The polynucleotide of the invention can be included in an expressionconstruct such as a vector, plasmid, virus/phagemid, artificialchromosome, cosmid, and further constructs known to the skilled personin order to provide for expression of the sequence of the mutant orhomologue of the invention. Techniques for modifying nucleic acidsequences for insertion into a vector e.g. by utilizing recombinant DNAmethods are also well-known in the art. Generally, an expression vectorcomprises the polynucleotide to be expressed, which is operably linkedto one or more control sequences (e.g., promoter, transcriptional stopsignal, translational stop signal, etc.) capable of directing theexpression of the polypeptide in the desired host cell. The promoter canbe an inducible or constitutive, general or cell specific promoter.Preferred examples of cell specific promoters are the dat-1 promoterfragment of the dopamine transporter gene core promoter and the humansynapsin-1 gene promoter. The selection of promoters, vectors and otherelements is a matter of routine design within the level of ordinaryskill in the art and many different such control sequences are describedin the literature and available through commercial suppliers. Generally,the choice of the vector will typically depend on the choice of the hostcell into which the vector will be introduced. Preferred expressionvectors for use in the present invention are the pT7-7, the C. elegansexpression vector pPD115.62, the GAL4-responsive pUAST expressionvector, and the AAV-1/2 mosaic serotype viral vector or derivativesthereof.

In a further aspect, the invention relates to a cell comprising thepolynucleotide or expression vector described above.

The polynucleotide or expression vector may be introduced into cells byvarious ways, e.g., using a virus as a carrier or by transfectionincluding e.g. by chemical transfectants (such as Metafectene,Lipofectamine, Fugene, etc.), electroporation, calcium phosphateco-precipitation and direct diffusion of DNA. Suitable transfectiontechniques are known to the skilled person and the method of choice willvary depending on the host cell to be transfected. Transfection of acell may yield stable cells or cell lines, if the transfectedpolynucleotide or expression vector is integrated into the genome, or byusing episomal replicating plasmids, i.e. that the inheritance of theextrachromosomal plasmid is controlled by control elements that areintegrated into the cell genome. In addition, unstable (transient) cellsor cell lines, wherein the transfected DNA exists in an extrachromosomalform can be produced.

The expression vector may further comprise a selectable marker, whichprovides for positive selection of transfected cells, i.e. transfectedcells exhibit resistance to the selection and are able to grow, whereasnon-transfected cells generally die. Examples of selective markersinclude puromycin, zeocin, neomycin (neo) and hygromycin B, which conferresistance to puromycin, zeocin, aminoglycoside G-418 and hygromycin,respectively. However, other selection methods known to the skilledperson may also be suitable.

The cell may be maintained and cultured at an appropriate temperatureand gas mixture (typically, 37° C., 5% CO₂), optionally in a cellincubator as known to the skilled person. Culture conditions may varyfor each cell type, and variation of conditions for a particular celltype can result in different phenotypes. Furthermore, recipes for growthmedia can vary in pH, glucose concentration, growth factors and thepresence of further suitable nutrient components. Growth media arecommercially available, or can be prepared according to compositions,which are for example obtainable from the American Tissue CultureCollection (ATCC). Growth factors used for supplement media are oftenderived from animal blood such as calf serum, but also other probablycell specific growth factors may be enclosed. Additionally, antibioticsmay be added to the growth media to prevent undesired microbial growth.More specifically, cell culturing is further exemplified in the Examplessection.

In general, the cell may be any kind of cell. For the expression andpurification of the mutant or homologue of the invention, a prokaryoticcell may be used, such as the E. coli strain BL21. However, for otherpurposes, including the methods described herein, a eukaryotic cell orcell line may be used. Cell lines which may be used in the invention arecommercially available from culture collection such as the ATCC or fromother commercial suppliers.

In general, the cell may exogenously express the mutant alpha-synuclein,i.e. the polynucleotide or expression vector encoding the mutantalpha-synuclein or homologue has been introduced into the cell. Thus,the mutant-alpha-synuclein or homologue according to the invention maybe derived from the same species or a different one than the cell.Optionally, a cell may endogenously express alpha-synuclein according toSEQ ID NO: 1, or a homologue thereof as defined above, i.e. thealpha-synuclein homologue is naturally expressed in the cell. In thatcase the cell may be obtained by genetic engineering of the endogenousgene expressing wild-type alpha-synuclein.

In a preferred embodiment, the cell is a yeast cell or an invertebratecell, preferably a cell of C. elegans or D. melanogaster, or wherein thecell is a vertebrate cell, preferably a mammalian cell, more preferablya mouse, a rat, or a primate cell, in particular a non-human embryonicstem cell. It is noted that those cells or cell lines, particularlyhuman embryonic stem or germline cells, are excluded, which are notsubject to patentability under the respective patent law orjurisdiction.

Dopaminergic neuronal cells are neurons whose primary neurotransmitteris dopamine, which has many functions in the brain, including importantroles in behavior and cognition, motor activity, motivation and reward,inhibition of prolactin production (involved in lactation), sleep, mood,attention, and learning. Dopaminergic neuronal cells are mainly presentin the ventral tegmental area of the midbrain, substantia nigra parscompacta, and arcuate nucleus of the hypothalamus. As indicated above,symptoms of Parkinson's disease result from the loss of dopaminergiccells in the region of the substantia nigra pars compacta. Their lossleads to alterations in the activity of the neural circuits within thebasal ganglia that regulate movement. In general, it is considered thatsymptoms appear when 80% of these neurons are lost leading to ahypokinetic movement disorder. Accordingly, in another preferredembodiment, the cell according to the invention is a dopaminergicneuronal cell.

In a further aspect, the invention provides a non-human animalcomprising the cell according to the invention, wherein the non-humananimal is an invertebrate, preferably C. elegans, or D. melanogaster, orwherein the non-human animal is a vertebrate, preferably a mammal, morepreferably a mouse, a rat, or a primate.

Also, a non-human animal comprising the cell according to the invention,wherein the non-human animal is an invertebrate, preferably C. elegans,or D. melanogaster, or wherein the non-human animal is a mammal iscontemplated. More specifically, the invention contemplates a non-humananimal comprising a dopaminergic neuronal cell according to theinvention.

In general, the non-human animal may be any animal other than a human.However, some model organisms, which may preferably be used in theinvention, have gained particular attention in research. Amonginvertebrates, these are Caenorhabditis elegans, Arbacia punctulata,Ciona intestinalis, Drosophila, usually the species Drosophilamelanogaster, Euprymna scolopes, Hydra, Loligo pealei, Pristionchuspacificus, Strongylocentrotus purpuratus, Symsagittifera roscoffensis,and Tribolium castaneum. Among vertebrates, these are several rodentspecies such as guinea pig (Cavia porcellus), hamster, mouse (Musmusculus), and rat (Rattus norvegicus), as well as other species such aschicken (Gallus gallus domesticus), cat (Felis cattus), dog (Canis lupusfamiliaris), Lamprey, Japanese ricefish (Oryzias latipes), Rhesusmacaque, Sigmodon hispidus, zebra finch (Taeniopygia guttata),pufferfish (Takifugu rubripres), african clawed frog (Xenopus laevis),and zebrafish (Danio rerio). Among vertebrates, mammals are particularlypreferred. Also preferred are non-human primates, i.e. all species ofanimals under the order Primates that are not a member of the genusHomo, for example rhesus macaque, chimpanzee, baboon, marmoset, andgreen monkey. However, these examples are not intended to limit thescope of the invention. It is noted that those animals are excluded,which are not likely to yield in substantial medical benefit to man oranimal and which are therefore not subject to patentability under therespective patent law or jurisdiction. Moreover, the skilled person willtake appropriate measures, as e.g. laid down in international guidelinesof animal welfare, to ensure that the substantial medical benefit to manor animal will outweigh any animal suffering.

Such a non-human animal may serve as a model system forsynucleinopathies (sometimes also named alpha-synucleinopathies), suchas dementia with Lewy bodies (DLB), Parkinson disease, Parkinson'sdisease with dementia (PDD), multiple system atrophy and the Lewy bodyvariant of Alzheimer's disease. Thus, synucleinopathies describe a groupof diseases which have all a common involvement of alpha-synuclein.

In a further aspect, the invention provides a method for identifying asubstance that prevents or reduces toxicity of alpha-synuclein to a testcell, the method comprising:

-   -   (a) culturing the cell according to the invention;    -   (b) contacting the cell with a test substance; and    -   (c) comparing the cell viability of the cell subjected to        step (b) with the cell viability of a corresponding cell        subjected to step (a), but not step (b);

wherein an increase in the cell viability of the cell subjected to step(b) compared to the cell viability of a corresponding cell subjected tostep (a), but not step (b), is indicative of the capability of thesubstance to prevent or reduce toxicity of alpha-synuclein.

The test substance may be provided in the form of a chemical compoundlibrary including a plurality of chemical compounds which have beenassembled from any of multiple sources, such as chemically synthesizedmolecules and natural products, or have been generated by combinatorialchemistry techniques. They may have a common particular structure or maybe compounds of a particular creature such as an animal. In the contextwith the present invention the test substance may comprise smallmolecules, proteins or peptides.

The cell viability may be determined as described above using thewater-soluble tetrazolium salt (WST) colorimeteric assay for determiningmitochondrial dehydrogenase activity according to the protocol of themanufacturer. There are other assays known in the art, which are alsocommercially available as kits for determining the cell viability, suchas kits comprising a fluorescence-dye labelled anti Annexin V antibody(abcam, BD, BIOMOL). Specifically late apoptotic cells may be detectedby adding ethidium bromide to a sample of the cells following flowcytrometric analysis. Furthermore, cell proliferation, which is anothermarker of cell viability, may be determined by an XTT assay (Roche). Theperson skilled in the art will know which assay to choose depending onthe cell which is applied in the method of the invention.

In the context of the present invention, the cell viability of the cellsubjected to step (a), but not step (b) of less than 90%, less than 80%,less than 70%, in particular less than 65%, less than 60%, less than55%, less than 50%, less than 45%, less than 40%, less than 35%, lessthan 30%, less than 25%, less than 20%, less than 15%, less than 10%, orless than 5% in comparison to the cell which has been subjected to step(a) and (b) is indicative for an increase in the cell viability.

Further, the invention provides a method for identifying a substancethat prevents or reduces toxicity of alpha-synucleinto a test animal,the method comprising:

-   -   (a) providing a non-human animal according to the invention;    -   (b) administering a test substance to the non-human animal; and    -   (c) comparing the result of a suitable test selected from the        group consisting of tests for the locomotion, sleeping behavior,        circadian rhythm, behavior, lifespan, retinal degeneration        and/or the neuronal degeneration for the non-human animal        subjected to step (b) with the result of the same test for a        corresponding non-human animal subjected to step (a), but not        step (b);

wherein a difference in the result of said test for the non-human animalsubjected to step (b) in comparison to the result of said test for thecorresponding non-human animal subjected to step (a), but not step (b),is indicative of the capability of the substance to prevent or reducetoxicity of alpha-synuclein.

The skilled person will know which test is suitable, depending on thechoice of the non-human animal.

It is well-known in the art that synucleinopathies affect thelocomotion. Thus, one suitable test to identify whether a substance iscapable to prevent or reduce toxicity of alpha-synuclein is a test forthe locomotion. The tests will vary depending on the choice of thenon-human animal. For example, if the non-human animal was a fly, suchas Drosophila melanogaster, one suitable well-known test would be theclimbing assay. In this assay the flies to be tested are placed in anapparatus in a black case, containing a bottom vial and an invertedupper vial, wherein they are assayed for their ability to reach theupper vial from the bottom vial in twenty seconds. Since flies generallyget attracted towards light, a light source is provided at the top ofupper vial with the help of two light emitting diodes. This type of setup provides a directionality and motivation for the flies to climb up.Other tests for the locomotion of an animal are known in the art.

If the non-human animal(s) subjected to step (b) has/have an improvedlocomotion compared to the non-human animal(s) subjected to step (a),but not step (b), this is indicative for the capability of the substanceto prevent or reduce toxicity of alpha-synuclein. In the context of theabove example of a test for locomotion, a higher percentage of the fliessubjected to step (b) which exhibit the ability to reach the upper vialin twenty seconds compared to the flies which have been subjected tostep (a), but not step (b), under the same conditions is indicative foran improved locomotion.

Other symptoms of synucleinopathies, such as Parkinson's disease, aresleep disturbances, characterized by excessive daytime somnolescence,initial, intermediate, and terminal insomnia, and disturbances in REMsleep. Somnolescence describes a state of near-sleep, a strong desirefor sleep, or sleeping for unusually long periods. It has two distinctmeanings, referring both to the usual state preceding falling asleep,and the chronic condition referring to being in that state independentof a circadian rhythm. Thus, tests for circadian rhythm are closelyrelated to tests for sleeping behaviour and may be tested in the sameway. Insomnia is a symptom of a sleeping disorder characterized bypersistent difficulty falling asleep or staying asleep despite theopportunity and is typically followed by functional impairment whileawake.

Thus, one suitable test to identify whether a substance is capable toprevent or reduce toxicity of alpha-synuclein is a test for the sleepingbehavior or circadian rhythm. Again, tests will vary and the skilledperson will know which test to apply, depending on the choice of thenon-human animal. For example, if the non-human animal was a fly, suchas Drosophila melanogaster, one suitable test would be a sleep assay.Briefly, fly embryos are collected in 2 hour window periods, and aregrown under LD 12:12 at 25° C. before the eclosion. Males are collectedfrom the progeny and aged with equal population density under LD 12:12at 25° C. After 25-30 days, locomotor activity of the aged flies isrecorded in LD by the Drosophila Activity Monitoring (DAM) system(Trikinetics, Waltham, Mass.) as described in (Hendricks et al. NatNeurosci 4, 1108-15 (2001)). Sleep is measured as bouts of 5 min ofinactivity, using a moving window of 1 min intervals. Average boutlength (ABL) is calculated from the sum of sleep bouts of all lengths(in minutes) divided by the total number of sleep bouts. Furthermore, anactivity index can be calculated by dividing total daily activity by thetotal wake time of the flies subjected to step (b) and the fliessubjected to step (a), but not step (b).

If the non-human animal(s) subjected to step (b) has/have decreasedsleep disturbances compared to the non-human animal(s) subjected to step(a) but not step (b), this is indicative for the capability of thesubstance to prevent or reduce toxicity of alpha-synuclein. In thecontext of the above example of a test for sleeping behavior, a longersleep or a decreased activity index of the flies subjected to step (b)compared to the flies which have been subjected to step (a) but not step(b), under the same conditions is indicative for decreased sleepdisturbances.

Other suitable tests to identify whether a substance is capable toprevent or reduce toxicity of alpha-synuclein are tests commonlyreferred to as tests for behavior. Examples for tests for behaviorencompass tests for cognition (e.g., voluntary and involuntary motorresponses), tests for memory (e.g., short-term memory, proceduralmemory), and social tests. However, many other tests for behavior areknown in the art as well. Also, tests will vary and the skilled personwill know which test to apply, depending on the choice of the non-humananimal. For example, if the non-human animal was a nematode, such as C.elegans, one suitable test would be an assay for the response to thepresence of food. Typically, healthy animals will slow down theirmovement in order to feed more efficiently as a dopamine-controlledbehavior. Therefore, this behavior allows to directly assess thefunctional integrity of dopaminergic neurons in C. elegans. Forbehavioral analysis wellfed adult animals are transferred to the centerof an assay plate with or without food as described previously (Brenner,S. Genetics 77, 71-94 (1974)). After an initial time of adjustment for 5min the movement was assayed by counting body bends over a one mininterval with three repetitions per animal. The slowing rate wascalculated and defined as the percentage of locomotion on food ascompared to the locomotion on plates without food.

If the non-human animal(s) subjected to step (b) show(s) a more similarbehavior compared to healthy animals in comparison to the non-humananimal(s) subjected to step (a) but not step (b), this is indicative forthe capability of the substance to prevent or reduce toxicity ofalpha-synuclein. In the context of the above example of a test forbehavior, a higher percentage of worms that slow down locomotion of theworms subjected to step (b) compared to the worms which have beensubjected to step (a) but not step (b), under the same conditions isindicative for a more similar behavior compared to healthy animals.

Tests for lifespan are well known to the skilled person and comprise allkind of techniques for determining the lifetime of an animal startingfrom birth or hatching (optionally also eclosion) and ending with thedeath of the non-human animal.

Tests for determining retinal degeneration are well known in the art,and the skilled person will know how to choose a suitable test.

Finally, the invention provides a method for identifying a substancethat prevents or reduces toxicity of alpha-synuclein to a test animal,the method comprising:

-   -   (a) providing a non-human animal according to the invention and        subjecting said non-human animal to a suitable test selected        from the group consisting of tests for the locomotion, sleeping        behavior, circadian rhythm, behavior, retinal degeneration        and/or the neuronal degeneration;    -   (b) administering a test substance to the non-human animal and        subjecting the animal to the same test; and    -   (c) comparing the result obtained in step (b) with the result        obtained in step (a);

wherein a difference in the result of said test for step (a) incomparison to step (b) is indicative.

DESCRIPTION OF THE FIGURES

FIG. 1. Structure-based design of alpha-S mutants.

A, Functional domains of alpha-S. Familial mutants (E46K, A53T, A56P)and design mutants (A30P, A76P) are labeled along the sequence. Regionsinvolved in beta-sheet formation in the fibril are marked (purple). B,Superimposed contour plots of the ¹H—¹⁵N HSQC spectra of wt (black) andTP (red) alpha-S in the free state at 15° C. Affected resonances arelabeled. Mutated residues are shown in red. C, Circular dichroismspectra of A56P alpha-S (yellow), TP alpha-S (red) and wt alpha-S(black) in solution (top panel) and in the presence of sodium dodecylsulfate micelles (bottom panel). Mutations do not strongly change theoverall secondary structure of alpha-S in the two states. D, Diffusionproperties of A56P alpha-S (circles), TP alpha-S (long dashes) and wtalpha-S (black short dashes) as observed by NMR signal decays in pulsedfield gradient measurements at 15° C. 1,4-dioxane (dark blue) was usedas internal standard. The fact that the diffusion properties are notsubstantially different in wt and mutant alpha-S indicate that theoverall shape averaged over the ensemble of conformations is not changedby the mutations.

FIG. 2. Point mutations delay formation of amyloid fibrils of alpha-S.

A, Fibril formation of wt alpha-S (black), A56P alpha-S (yellow) and TPalpha-S (red) followed by Thioflavin T fluorescence. B, Consumption ofmonomeric wt alpha-S (black), A53T (cyan) A30P alpha-S (purple), A56Palpha-S (yellow) and TP alpha-S (red) monitored by 1D ¹H NMRspectroscopy. Drop in signal intensity is due to formation of highermolecular weight aggregates not detectable by solution-state NMR. Errorswere estimated from three independent aggregation assays. C, Dynamiclight scattering of A56P alpha-S (yellow) and TP alpha-S (red). Datapresented here are a representative of 30 acquisitions. D, Pre-fibrillaraggregates of TP alpha-S seed fibril formation of wt alpha-S. In anequimolar mixture of pre-aggregated TP alpha-S with monomeric wt alpha-Sthe lag time of aggregation is decreased (light blue) when compared towt monomer alone (black), whereas in an equimolar mixture of monomericTP alpha-S and monomeric wt protein the lag time is increased (darkblue). Aggregation behaviour of TP alpha-S alone is shown in red. Errorbars in A, B and D represent mean±standard deviation of threeindependent experiments. E, Recognition of a mixture of oligomers andmonomers of TP alpha-S (O/M) but not monomeric TP alpha-S (M) onnitrocellulose membrane by the A11 antibody. Anti-alpha-S antibody showscomparable attachment to both monomer and oligomer. F, Aggregateformation and toxicity in HEK293T cells: alpha-S aggregation wasvisualized by adding the six amino acids of a PDZ binding domain to theC-terminus and coexpressing the corresponding PDZ domain fused to EGFP(ctrl, PDZ-EGFP alone). Cells with more than one aggregate(“aggregation”, left axis, clear bars) and preapoptotic cells(“toxicity”, right axis, hached bars) were counted 24 h aftertransfection. Bars represent percentages of all EGFP-positive cells(mean±SEM, n=5 independent experiments). Significances are depicted withrespect to ctrl (n.s., not significant; **, p<0.01, One-way ANOVA andDunnet's posthoc test). G Fibril formation of wt alpha-S (black), A30P(purple), A53T (blue), A56P alpha-S (yellow) and TP alpha-S (red, alsoshown in Inset A at a different scale) followed by Thioflavin T (ThT)fluorescence emission intensity. Inset B demonstrates ThT intensityvalues of all but TP variants, each of them normalized by the maximalvalue observed along their aggregation reaction.

FIG. 3. Structure-based design mutants of alpha-S cause movementdisorders, sleep impairment and reduced life span of C. elegans andDrosophila.

A, ‘Basal slowing response’ of C. elegans expressing different alpha-Svariants in dopaminergic neurons. For each alpha-S variant expressed atleast two independent transgenic lines were tested (n=40-50 animals pertrail, 3 trails). The slowing rate corresponds to the average decreasein movement (body bends/min) for animals placed in food as compared toanimals without food. Animals expressing only EGFP in dopaminergicneurons are shown as control. The error bar correspond to the standarderror of the mean (SEM) and the significance values of the ANOVA testare indicated: * p<0.05; ** p<0.01; *** p<0.001. B, Climbing assay onflies with corresponding genotypes. Climbing index, percentage of 25-30day old flies that could reach the top chamber in a fixed amount of time(n=35-50 for each group). C, Survival curves of flies expressingdifferent variants of alpha-S and LacZ. A56P alpha-S and TP alpha-Scurves are significantly different from wt alpha-S (Logrank Test:P<0.0217 for wt alpha-S versus A56P alpha-S, and P<0.0001 for wt alpha-Sand TP alpha-S. n=350-400 for each genotype). D) Averaged sleep profilesof wt alpha-S (black), A56P alpha-S (yellow) and TP alpha-S (red)(shading represents lights off). E) Changes in the average length ofsleep bouts (n=40-50 for each genotype). Where errors are shown, theyare s.e.m. Significance was determined by one-way analysis of variancefollowed by Dunnett's Multiple Comparison test. *P<0.05; **P<0.01; n.s.non significant in comparison with wt alpha-S, P>0.05.

FIG. 4. Neurotoxicity of structure-based design mutants of aS inmammalian neurons, C. elegans and Drosophila.

(A) Structure-based design variants in rat primary neurons. Left panel:WST assay of cortical neurons transduced by AAV-EGFP, AAV-alpha-S-wt,AAV-alpha-S-A30P, AAV-alpha-S-A53T, AAV-alpha-S-A56P and AAV-alpha-S-TP,respectively. Mitochondrial dehydrogenase activity measured aftertransduction with respective alpha-S mutants is shown as percentage ofactivity measured after AAV-EGFP transduction (n=30). Middle panel:Neuronal cell loss quantified by NeuN immunocytochemistry. Numbers ofNeuN immunoreactive cells counted after transduction with respectivealpha-S mutants is shown as percentage of numbers counted after AAV-EGFPtransduction (n=15). Right panel: Degeneration of dopaminergic midbrainneurons quantified by TH immunocytochemistry. Numbers of THimmunoreactive cells counted after transduction with respective alpha-Smutants is shown as percentage of numbers counted after AAV-EGFPtransduction (n=12). Data are shown as mean+/−SEM. In all casessignificance was determined by one-way ANOVA analysis of variancefollowed by Dunnett's posthoc test *P<0.05; **P<0.01. (B) C. elegansexpressing red fluorescent protein mCherry and wt alpha-S (upper leftpanel) or TP alpha-S (lower left panel) in the cephalic (CEP) andanterior deirid (ADE) dopaminergic neurons in the head. Right panel:degeneration of dendritic processes induced by expression of αS indopaminergic neurons. Two independent transgenic lines are shown peralpha-S variant and 78-80 animals we analyzed per line. (C) Whole-mountimmunostaining of fly brains. Images are maximum projections of severalconfocal sections in the z-plane. (D) Quantitative analysis ofdopaminergic neuron numbers in the dorsomedial (DM) and dorsolateral(DL) cluster in brains of 2 day (young) and 29 day (adult) old flies.Values represent mean+/−SEM. Asterisks indicate that the difference indopaminergic neuron numbers was statistically significant. For 2 day oldflies, no statistically significant difference was observed in numbersof dopaminergic neurons. Expression levels of different alpha-S variantswere comparable (FIG. 23).

FIG. 5. Monomeric forms of design variants of aS remain disordered insolution.

(A) Superimposed contour plots of the ^(J)H—¹⁵N HSQC spectra ofmonomeric wt (black), A56P (yellow) and TP (red) alpha-S in solution at15° C. Influenced resonances are labeled. Mutated residues are marked(red). (B) Mean weighted ^(J)H—¹⁵N chemical shift differences between wtand A56P aS (yellow) and between wt and TP aS (red) in the free State(calculated from [(A5 ^(J)H)²+(A5¹⁵N)²/25]^(1/2))/2]).

FIG. 6. Design mutations do not prohibit the interaction of alpha-S withnegatively charged SDS micelles.

¹H—¹⁵N HSQC spectra of free wt (A), A56P (B) and TP (C) alpha-S. ¹H—¹⁵NHSQC spectra of wt (D), A56P (E) and TP (F) alpha-S in the presence ofSDS micelles at 40° C. and pH 7.4. An increase in resonance dispersionwith respect to free spectra is observed upon addition of the micellesfor all three variants.

FIG. 7. Electron microscopy analysis of the aggregation of wt, A56P andTP alpha-S.

Electron micrographs shown are representative pictures for triplicates.Scale bars correspond to 200 nm, except wt alpha-S 56h and TP alpha-S21h, where they correspond to 500 nm and 100 nm, respectively. Oligomerswere observed in all three samples early on in the aggregation process.Mature fibrils were observed after about 50 h for wt alpha-S, afterabout 100 h for A56P alpha-S and no traces of mature fibrils weredetected for TP alpha-S in a period of two weeks. Total proteinconcentration in each sample was 100 μM.

FIG. 8. Morphology of soluble oligomers formed by alpha-S variants.

(A) Electron micrographs of soluble oligomers formed by alpha-Svariants. The protein concentration was 100 μM and samples wereaggregated for 12 h at 37° C. and 200 rpm. Scale bars correspond to 200nm. (B) Dynamic light scattering of alpha-S variants. Data presentedhere are a representative of 30 acquisitions of 10 s.

FIG. 9. Thioflavin T measurements of the alpha-S variants.

From left to right: Wt (black), A30P (purple), A56P (yellow), A76P(green), A30PA56P (blue), A30PA76P (magenta).

FIG. 10. High Resolution solid-state NMR of late stage aggregates formedby alpha-S variants.

(A) Water accessibility of aggregates as probed by solid-state NMR. A 3ms Gaussian pulse and a T₂ filter containing two delays of 1 ms wereused for selective water excitation. The cross polarization contact timewas set to 700 μs. (B) Superposition of 2D ¹³C/¹³C correlation spectraof U-[¹³C,¹⁵N] A56P alpha-S (yellow) and of wt alpha-S (black).Correlations absent in the A56P mutant are underlined. Assignmentscorrespond to values obtained for the A form of wt alpha-S as reportedin (Heise et al., 2005). (C) Superposition of 2D ¹³C/¹³C correlationspectra of U-[¹³C,¹⁵N] A56P alpha-S (yellow) and of U-[¹³C,¹⁵N] TPalpha-S (red). In (B) and (C), homonuclear mixing was achieved using aproton driven spin diffusion time of 20 ms (A56P) and 50 ms (TP),respectively. (D) 2D ¹H/¹³C ¹H-T₂-filtered HETCOR spectrum of A56Palpha-S. The spectrum was recorded at a magnetic field strength of 14 T,with a spinning speed of 8.33 kHz, at a sample temperature of 0° C. TheT₂ filter delay was 2×175 μs, the contact time was 3 ms. The spectrumwas recorded without homonuclear decoupling during t₁, 160 t₁ incrementsand 128 scans per slice. (E) ssNMR-based secondary structure analysisfor wt and mutant alpha-S. Row 1 and 2 correspond to wt data reportedpreviously (Heise et al., 2005). Hashed rectangles relate to proteinregions in which beta-strands are lost compared to wt alpha-S or, in thecase of TP, exhibit strong dynamics/disorder. Arrows relate tobeta-strands that are preserved in A56P. Mutation sites are indicated byrectangular boxes, suggesting that A⇄P mutation leads to partial (A56P)or almost complete (TP) suppression of beta-strand formation in alpha-S.

FIG. 11. Targeting alpha-S variants to an identical genomic locationensures comparable expression levels in transgenics.

A, Schematic representation of the Drosophila transgenesis based onφ-C31 mediated recombination. B, In all transgenic animals expressingalpha-S mutants (lanes 3, 4, 5, 6), single fly PCR using a alpha-Sforward primer and a genomic reverse primer (depicted in a—genomic DNAafter the integration event) shows that integration has indeed occurredat the desired attP landing site in the 3R-86Fb genomic region. Ascontrols, flies carrying an empty-attP landing site at 3R-86Fb (lane 2)and flies carrying lacZ insertion at the same location (lane 7) wereused.

FIG. 12. Sleep profile of 25-30 day old flies.

expressing a, wt alpha-S (black) and lacZ control flies (blue) b, wtalpha-S (black), A53T alpha-S (cyan) and A56P alpha-S (yellow) under theddc-GAL4 driver.

FIG. 13. Total sleep and total activity of flies expressing alpha-Svariants.

a, Total sleep per day in flies expressing wt alpha-S (black), TPalpha-S (red), A56P alpha-S (yellow), A53T alpha-S (green) and lacZcontrol (blue). Differences in total sleep between flies expressingalpha-S mutants are not significant according to one-way ANOVA analysis(P>0.05). b, Activity index of alpha-S variants. The ‘Activity Index’was calculated by dividing total daily activity (number of beamcrossings per day) by the total wake time for each genotype. Accordingto one-way ANOVA analysis (P>0.05) followed by the Tukey's multiplecomparison test differences are not significant; n=30-40 per genotype.

FIG. 14. alpha-Synuclein mutated in the C-terminus remains disordered.

¹H—¹⁵N HSQC nuclear magnetic resonance spectrum of M127A alpha-synucleinin solution. Resonance assignments are indicated.

FIG. 15. Toxicity assay using C-terminal mutants of alpha-synuclein inyeast.

Yeast cells were inoculated to an OD600 of 0.1 and incubated underalpha-synuclein inducing conditions (SC-ura/Galactose). Toxicity of thevarious alpha-synuclein mutants can be judged by the growth impairmentcompared to vector control.

FIG. 16. Movement disorder and neurotoxicity induced by design mutantsof alpha-synuclein in C. elegans.

A, ‘Basal slowing response’ of C. elegans expressing differentalpha-synuclein variants in dopaminergic neurons. For eachalpha-synuclein variant expressed at least two independent transgeniclines were tested (n=40-50 animals per trail). The slowing ratecorresponds to the average decrease in movement (body bends/min) foranimals placed in food as compared to animals without food. Animalsexpressing only EGFP in dopaminergic neurons are shown as control.

B, Degeneration of dendritic processes induced by expression ofalpha-synuclein in dopaminergic neurons. Two independent transgeniclines are show per alpha-synuclein variant and 78-80 animals wereanalyzed per line.

FIG. 17. Enhanced formation of soluble oligomers by αS variants.

(A) Electron micrograph of TP alpha-S solution in 50 mM HEPES, 100 mMNaCl, pH 7.4, 0.01% NaN3, incubated for 6 days at 37° C. while stirredat 200 rpm. The protein concentration was 0.8 mM, and the sample wasdiluted 8-fold by buffer before EM imaging. Scale bar corresponds to 500nm. (B) AFM image of TP alpha-S solution. Conditions identical to A).(C) Dynamic light scattering of alpha-S variants, incubated for 11 daysat the aggregation condition and then centrifuged briefly and thesupernatant was measured. Data presented are average of threemeasurements, each consisting of 20 acquisitions of 20 s. (D). UVabsorbance of the supernatant of aggregated alpha-S variants after 11days of incubation at the aggregation condition.

FIG. 18. Aggregate formation and toxicity in HEK293T cells.

(A) Real time PCR quantification of alpha-S mRNA extracted 24 h aftertransfection. Bars represent alpha-S mRNA relative to the ribosomal 18Ssubunit mRNA. (mean±SEM, n=3 independent experiments, no significantdifferent between different alpha-S variants) (B) Quantification of thewestern-blot bands (exemplified in E) no significant different betweenthe different alpha-S variants (mean±SEM, n=3 independent experiments).(C) Western-blot of transfected (24 h) HEK293T cells. Alpha-S includingthe PDZ binding domain has a molecular weight close to 19 kDa, PDZdomain fused to EGFP has a predicted molecular weight of 46 kDa andbeta-actin is close to 42 kDa. (D) Representative images of aggregates(top panel, arrowheads) and preapoptotic cell (lower panel). Scale baris 10 μm.

FIG. 19. TP alpha-S displays reduced aggregation propensity in vivo.

Ten-day old vulva muscles are show from transgenic animals expressingeither wt alpha-S A) or TP alpha-S B) fused to mYFP. Only wtalpha-S-mYFP leads to extensive fibrilar aggregates while TPalpha-S-mYFP remains diffusely distributed in the cytoplasm. (C) Theexpression levels of the alpha-S-mYFP fusion proteins are similar asshown by Western blot using anti alpha-S antibodies. Tubulin stainingserves as a loading control.

FIG. 20. Electron micrographs of alpha-S variants.

Electron micrographs of alpha-S variants (A: wt, B: A30P, C: A53T, D:A56P, E: TP) after 5 days of incubation at 37° C., 50 mM HEPES, 100 mMNaCl, pH 7.4 and 0.01% NaN₃, stirred at 200 rpm. The proteinconcentration was 0.8 mM. Scale bars correspond to 1000 nm for wt, A30Pand A53T and 500 nm for A56P and TP alpha-S.

FIG. 21. Circular dichroism spectra of alpha-S variants in the freestate (A) and when bound to SUVs formed by POPC:POPA (ratio 1:1) (B).

FIG. 22. Binding of αS variants to phospholipid vesicles.

Vesicles prepared from a 1:1 mixture of POPC and POPA were incubated for5 hours at room temperature with alpha-S variants at a mass ratio of250:1, and the mixture was separated by gel filtration chromatography ona Superose 6 10/300 GL column (GE healthcare). For comparison,lipid-free alpha-S variants (thin black line) were subjected to similarseparation.

FIG. 23. Expression levels of different alpha-S variants are comparablein (A) C. elegans, (B) rat neuronal cultures and (C) fruit flies. In(B), A53T alpha-S runs at a slightly higher molecular weight due to thepresence of the seven amino acid (DTYRYI) long epitope tag AU1.

EXAMPLES

In the following, the present invention is illustrated by figures andexamples which are not intended to limit the scope of the presentinvention.

Example 1

Design of Alpha-S Variants

A design of alpha-S variants that aims at the production of toxicspecies should keep the structural and functional properties of therespective multimers nearly constant. The design was based on theconformational properties of the alpha-S monomer in solution and thetopology of alpha-S fibrils known from previous solid-state nuclearmagnetic resonance (NMR) measurements (Lee et al., supra; Heise et al.Proc Nati Acad Sci USA 102, 15871-15876 (2005)). The genetic mutationA30P is located in a region of alpha-S that is statically disordered inamyloid fibrils (Heise et al., supra). To interfere with aggregation,the single proline mutation found in the genetic mutant were moved to aposition that is part of the beta-sheet rich core of alpha-S fibrils(FIG. 1A). The alanine residues 56 and 76 were selected as they arecharacterized by relatively large residual dipolar coupling values inthe soluble monomer, suggestive of a rigid nature (Lee et al., supra;Bertoncini et al., 2005).

Cloning, Expression, and Purification of Alpha-S Variants

pT7-7 plasmid encoding for human wt alpha-synuclein (alpha-S) was kindlyprovided by the Lansbury Laboratory, Harvard Medical School, Cambridge,Mass. A codon replacement was performed for residue Y136 (TAC to TAT)for codon usage concerns. The resulting construct was then used as thetemplate for mutagenesis reactions. Mutations were performed by usingthe QuickChange site-directed mutagenesis kit (Stratagene) and verifiedby DNA sequencing. Plasmids containing alpha-S variants were expressedin Escherichia coli BL21 (DE3) cells. Following transformation, cellswere grown in LB in the presence of ampicillin (100 μg/ml). Induction ofexpression was performed by 1 mM IPTG at 37° C. for five hours and cellswere then harvested by centrifugation. Cell lysis was performed by threeconsecutive freeze-thaw cycles that were followed by sonication. Themajority of the host cell proteins were then denatured by incubation ofthe crude extract at 95° C. for 20 min in a water bath and thesupernatant containing the soluble protein fraction was recovered bycentrifugation. Streptomycin sulfate was added to the supernatant to afinal concentration of 10 mg/ml and the mixture was gently rotated for15 minutes at 4° C. After centrifugation, the supernatant was collectedand ammonium sulfate was added slowly to a final concentration of 0.36g/ml. The solution was stirred for 15 minutes at 4° C. and centrifuged.The resulting pellet was resuspended in 25 mM Tris-HCl (pH 7.7). Afterovernight dialysis against the same buffer, anion exchangechromatography was performed at room temperature on a AKTA Basic system(Amersham Pharmacia Biotech) by using a POROS 20 HQ (Pharmacia Biotech)column. The protein was eluted in a linear NaCl gradient. Subsequentsize-exclusion chromatography over HiLoad Superdex 75 (PharmaciaBiotech) ensured a high purity of alpha-S samples. Finally, the proteinwas dialysed overnight against buffer. The protein concentration wasestimated from the absorbance at 280 nm using an extinction coefficientof 5960 M-1 cm-1. For production of 15N-labeled proteins, M9-minimalmedium supplemented with 15NH4Cl (Cambridge Isotope Laboratories) wasused. For double labeled solid-state samples, 13C-Glucose (CambridgeIsotope Laboratories) was also added to the M9-minimal medium.

The following alpha-S variants were generated: the genetic mutant A30P,the single-proline design mutants A56P, A76P, the double mutantsA30PA56P and A30PA76P, and the triple mutant A30PA56PA76P (TP alpha-S),respectively. Based on the known beta-breaking propensity of prolineresidues, these mutations are expected to cause an increasingly strongdelay in aggregation.

Nuclear Magnetic Resonance (NMR) Spectroscopy

NMR samples contained ˜0.2 mM 15N-labeled wt or mutant alpha-S in 50 mMNa-phosphate buffer, 100 mM NaCl at pH 7.4 and 90% H₂O/10% D₂O. Theexperiments were recorded on a Bruker Avance 600 MHz NMR spectrometer.The temperature was set to 15° C. unless otherwise stated. Dataprocessing was performed using the software packages Topspin (Bruker)and Sparky (Goddard, T. D., Kneller, D. G., University of California,San Francisco). For chemical shift analysis, ¹H—¹⁵N Heteronuclear SingleQuantum Coherene (HSQC) 2D spectra were recorded. Mean weighted ¹H—¹⁵Nchemical shift differences were calculated according toΔδ={[(Δδ¹H)2+((Δδ¹⁵N)/5)2]1/2}/2. NMR experiments on SDS micelle-boundalpha-S were performed by addition of deuterated SDS directly to theprotein sample prior to the measurement to a final concentration of 40mM. Measurements were performed at 40° C.

Pulse field gradient NMR experiments were performed with the PG-SLED(pulse gradient stimulated echo longitudinal encode-decode) pulsesequence (Jones et al. Journal of Biomolecular NMR 10, 199-203 (1997)).Sixteen one dimensional ¹H spectra were collected as a function ofgradient strength varying between 2% and 95% of its maximum value.Acqusitions were performed using 16000 complex data points with 32 scansper increment and a relaxation delay of 3 s. Samples contained ˜0.2 mMunlabeled wt or mutant alpha-S in deuterated 50 mM Na-phosphate buffer,100 mM NaCl at pH 7.4. As an internal hydrodynamic radius standard andviscosity probe, 0.1% 1,4-dioxane was included. After baselinecorrection, the decay in the intensity of the signals from the aliphaticregion was fitted as a function of gradient strength (g) to the equationf(g)=Ae^(−d(g^2)).

To follow the consumption of monomeric alpha-S by NMR, 0.5 ml samplescontaining 0.1 mM alpha-S in 50 mM Na-phosphate, 100 mM NaCl, 0.1% NaN₃,pH 7.4 and 90% H₂O/10% D₂O were incubated at 37° C. and stirred by 5×2mm stirring bars inside a standard NMR tube. At appropriate timeintervals, 1D ¹H spectra were measured. Spectra were baseline correctedand the decay in signal intensity was plotted as a function of time.

Circular Dichroism (CD) Spectroscopy

Far UV-CD measurements were performed on a Chirascan (AppliedPhotophysics, UK) circular dichroism spectrometer, using a proteinconcentration of 10 μM in 50 mM Naphosphate, 100 mM NaCl, pH 7.4 in aquartz cuvette with 0.1 cm light-path. In case of SDS micelle samples, aconcentration of 40 mM SDS was added. Recordings were performed over therange of 190-250, with 0.5 nm steps and 2 seconds per point. Thespectral bandwidth was 1 nm. Each experiment was repeated at leasttwice. Baseline correction was performed with the suitable buffer. Datawere expressed as mean residue ellipticity (degree cm² dmol⁻¹).

Binding of aS Variants to Small Unilamellar Vesicles (SUV)

1-Palmitoyl-2-Oleoyl phosphatidyl choline (POPC) and1-Palmitoyl-2-Oleoyl phosphatidic acid (POPA) were obtained from AvantiPolar Lipids. Vesicles were prepared from synthetic phospholipids in thefollowing ratios: POPC/POPA (1:1). The lipids were dissolved together ina 4 ml mixture of chloroform/methanol (1:1 vol/vol), followed by theevaporation of all solvents under a stream of N₂ gas and lyophilizedovernight. The resulting lipid film was hydrated in 20 mM TrisHCl (pH7.4), 100 mM NaCl or in 50 mM Na-phosphate buffer (pH 7.4), 100 mM NaClto obtain a total lipid concentration 12.5 mM. For preparation of SUV,the Suspension was bath sonicated at 37 kHz (4 times for 10 min with 5min breaks at room temperature) in a glass tube, and the SUV wereisolated by ultracentrifugation at 55,000 rpm in a Beckman TLA 100.3rotor for 2 hours at 298 K. The isolated SUV exhibited hydrodynamicdiameter of 20 nm±5 nm from dynamic light scattering. SUVs were mixedwith purified alpha-S variants at 250:1 mass ratio of phospholipid toprotein for 5 hours at room temperature, then subjected to gelfiltration on a Superose 6 10/300 GL column (GE healthcare) at a flowrate of 0.5 1/min. The peak volume (detected by UV at 280 nm) at theelution position of free synuclein was integrated and compared to thecorresponding peak volume obtained in the absence of SUV.

Results

The biophysical properties of wt and mutant alpha-S monomers were verysimilar. As expected, the chemical shift changes observed in ¹⁵N—¹Hheteronuclear single quantum coherence spectra by liquid-state NMRspectroscopy were small and restricted to the vicinity of the replacedresidues, indicating that mutation-induced structural changes are onlysubtle (FIG. 1B and FIG. 5). Moreover, secondary structure andhydrodynamic radius of the alpha-S monomer were not affected, asobserved with circular dichroism and pulsed field gradient spectroscopyassays, respectively (FIG. 1C,D). Furthermore, the alpha-S mutations donot prohibit the interaction with negatively charged micelles andsubsequent alpha-helix formation (FIG. 1C and FIG. 6), two features ofalpha-S that are potentially important for its cellular function.

Next we quantified the amount of alpha-S bound to SUVs, which wereformed by a 1:1 mixture of 1-Palmitoyl-2-Oleoyl phosphatidyl choline(POPC) and 1-Palmitoyl-2-Oleoyl phosphatidic acid (POPA). Gel filtrationof αS-SUV mixtures (phospholipid to protein mass ratio of 250:1)revealed that more than 95% of wt and A53T alpha-S are bound to SUVswhen using Tris buffer (Table 1 and FIG. 22). In case of A30P alpha-S,only 70±3% of the total protein was bound to POPC:POPA SUVs. A valuevery similar to that observed for A30P was obtained for A56P alpha-S. Inaddition, the affinity of TP alpha-S was only very slightly reducedcompared to A30P and A56P alpha-S, with 66±3% of SUV-bound protein(Table 1). When phosphate buffer was used instead of Tris, the overallamount of SUV-bound alpha-S was reduced, but relative differencesbetween different alpha-S variants were very similar(A53≈wt>A56P≈A30P≧TP) (Table 1). In addition, the content ofalpha-helix, which was detected by CD spectroscopy for the alpha-Svariants in the SUV-bound state, followed the amount of SUV-boundprotein (wt≈A53>A56P≈A30P≧TP) (FIG. 21 and Table 1).

TABLE 1 Alpha-helical content of alpha-S variants bound to POPC:POPASUVs. Sample Secondary structure predictions by K2D* (POPC/POPA 1:1)alpha-Helix (%) WT 52 A30P 31 A53T 46 A56P 35 TP 27 Determined using theK2D web server (http://www.embl-heidelberg.de/~andrade/k2d.htm).

Example 2

In Vitro Fibril Formation

Preparation of Alpha-S Aggregates

Recombinant human wt and mutant alpha-S solutions were dialysed against50 mM Na-phosphate buffer with 100 mM NaCl at pH 7.4 unless otherwisestated. To remove any potential seed prior to aggregation,ultracentrifugation was performed in a Beckman ultracentrifuge equippedwith TLA.100 rotor (Beckman Coulter) at 60000 rpm for 2 h at 4° C.Supernatant was filtered through 0.22 μm filter. Protein concentrationwas adjusted to 100 μM unless otherwise stated. 0.01% sterile filteredNaN₃ was included in the aggregation mixtures, which were then incubatedin glass vials at 37° C. with constant stirring at 200 rpm on amulti-position magnetic stirring device (Variomag Telesystem 15.40, H+PLabortechnic AG, Germany). For every experiment, at least triplicateswere prepared.

For seeded incubations, 200 μM solutions of TP alpha-S were incubatedfor five days at 37° C. prior to seeding. For electron microscopy, 50 mMHEPES 100 mM NaCl pH 7.4 was used. Without further purification orattempt to separate monomers from oligomers, the solution, whichcontained monomers and oligomers of TP aS, was added to wt monomericalpha-S at an equimolar ratio. As control, we performed an aggregationassay, in which monomeric TP alpha-S was added to monomeric wt alpha-Sat an equimolar ratio. Error bars in FIG. 2 D represent mean±Standarddeviation of three to four independent experiments.

NMR Spectroscopy

NMR spectroscopy was carried out as described in Example 1. The drop insignal intensity during aggregation is due to formation of highermolecular weight aggregates not detectable by solution-state NMR. Thus,the NMR signal intensity remaining during the course of the aggregationallows estimation of the concentration of monomeric protein.Simultaneously performed EM measurements, which were performed in theearly stages of the aggregation, only showed small oligomeric speciesand no amyloid fibrils. In addition, no increase in ThioT signalcompared to the monomeric protein was detected during the lag phase.Thus, the reduction of NMR signal intensity during the lag phase offibril formation allows estimation of the concentration of solubleoligomers. In case of the NMR aggregation assay performed for TP alpha-Sat a concentration of 0.1 mM (FIG. 18C), no amyloid fibrils weredetected during the complete time course of the experiment, indicatingthat the reduction in signal intensity is solely due to formation ofsoluble oligomers. Errors in the estimation of the oligomerconcentration depend on the basis of the signal-to-noise ratio in theNMR spectra and are determined from the variation observed in threeindependently performed aggregation assays. In case of TP alpha-S, theywere ±2%.

Thioflavin T (ThioT) Fluorescence Measurements

Aliquots (5 μl) were withdrawn from alpha-S incubations and added to 2ml of 5 μM ThioT in 50 mM Glycine-NaOH pH 8.2. Fluorescence measurementswere carried out on Cary Eclipse Spectrofluorometer (Varian) using 3.5ml quartz cuvettes (Hellma, Germany) with a path length of 1 cm.Fluorescence emission spectra were recorded from 465 to 600 nm, usingexcitation wavelength of 446 nm, an integration time of 0.1 second, andboth excitation and emission bandwidths of 10 nm. Kinetic aggregationtraces were generated from time traces of ThioT fluorescence intensityat 482 nm and corrected for free ThioT fluorescence. Aggregation yieldswere normalized to the final values and the averaged data points werefitted to a sigmoidal equation. (Data was represented as mean±standarddeviation, n=3).

Dynamic Light Scattering (DLS)

To monitor the build-up of oligomeric intermediates of alpha-S, aliquotsof 15 μl were withdrawn from the aggregation mixture at different timeintervals and measurements were performed directly on a DynaPro Titaninstrument (Wyatt Technology) at 25° C. Data analysis was performed withthe built-in software DYNAMICS from 30 successful measurements. For thehigher concentration samples (0.8 mM), 10 μl aliquots were taken at theend of incubation (11 days), then diluted 8-fold with the buffer andcentrifuged at 14000 rpm for 30 minutes, and the supernatant wasmeasured on a DLS machine. The same laser power was used for all alpha-Svariants. Data analysis was performed with the built-in softwareDYNAMICS from 30 successful measurements.

UV Spectroscopy

After 11 days of incubation of 0.8 mM alpha-S solutions in theaggregation condition, 10 μl aliquots were taken out and diluted 8-foldwith the buffer. Thereafter, the samples were centrifuged at 14000 rpmfor 30 minutes and the supernatant was investigated for apparent UVabsorbance in the 210-310 nm range.

Transmission Electron Microscopy (TEM)

For negative staining, a solution containing protein was applied toglow-discharged carbon coated grids and stained with 1% uranyl acetate.Images were taken in a Philips CM120 electron microscope (Philips Inc.)at a defocus of 2.3 μm using a TemCam 224A slow scan CCD camera (TVIPS,Gauting, Germany).

Atomic Force Microscopy (AFM)

A TP alpha-S solution (0.8 mM) in 50 mM HEPES, 100 mM NaCl, pH 7.4, with0.01% NaN3 was incubated at 37° C. with stirring at 200 rpm. An aliquotof 2 μl was diluted 8-fold in the above-mentioned buffer and 4 μl of thediluted sample were deposited on freshly cleaved mica. After drying inair for 1 hr, unbound sample and buffer were washed out with 100 μl ofdistilled water. The samples were imaged using an Asylum MFP3D AFMmachine, with a resonant frequency of about 100 kHz, a scan frequency of1 Hz, using silicone nitride tips.

Solid-State NMR Spectroscopy

For solid-state NMR measurements, 200 μM 13C- and 15N-labeled A56Palpha-S was incubated for two weeks and 200 μM 13C- and 15N-labeled TPalpha-S was incubated for four weeks at 37° C. and 200 rpm.Subsequently, alpha-S aggregates were recovered by centrifugation at60000 rpm for 2 h at 4° C. (TLA.100, Beckman ultracentrifuge).Two-dimensional NMR experiments were conducted on 14.1 T (¹H resonancefrequency: 600 MHz) and 18.8 T (¹H resonance frequency: 800 MHz) NMRinstruments (Bruker Biospin, Germany) equipped with 4 mmtriple-resonance (¹H, ¹³C, ¹⁵N) MAS probes. All experiments were carriedout at probe temperatures of 0° C. MAS rates were set to values thatfacilitate sequential correlations at longer mixing times, i.e., 9375 Hzat 600 MHz and 12500 Hz at 800 MHz. Resonance assignments for A56Palpha-S and a residue-specific analysis of beta-strands in alpha-Svariants was based on sequential (¹³C—¹³C) correlation data obtained atmixing times of 150 ms (data not shown).

Dot Blotting

Purified recombinant proteins were spotted onto nitrocellulose membrane.Blotting was performed using the conformation-specific A11 antibody(Invitrogen's Biosource). The amount of protein used for each spot was10 μg. In a parallel experiment, same samples were blotted using theanti-alpha-S antibody (BD Biosciences). The amount of the protein usedwas 1 μg.

Results

Despite the high structural resemblance of the monomeric proteins,fibril formation in vitro and in cells was dramatically reduced by thealpha-S mutations (FIG. 2 and FIG. 9). Whereas 0.1 mM of wt and A30P αSformed fibrils after about 20-30 hours as probed by thioflavin T (ThioT)fluorescence, A56P, A30PA56P and A30PA76P alpha-S had an approximatelyfive time longer lag phase (FIG. 2A and FIG. 9). In addition, theirfibril elongation rate was strongly reduced suggesting a reducedcooperativity of the transition. TP alpha-S did not show any fibrilseven after two weeks of incubation (FIG. 2A). However, electronmicroscopy and dynamic light scattering detected pre-fibrillar TPalpha-S aggregates already at an early stage of the aggregation process(FIG. 2 c and FIG. 7). A quantitative analysis of the NMR signal decayshowed that after 50 hours of incubation at 37° C. and 0.1 mM proteinconcentration the oligomeric intermediates constituted a 6% and 2%fraction of the protein mixture for A56P and TP alpha-S, respectively.In case of TP alpha-S, the oligomeric fraction increased to 4% after 160hours (FIG. 2B).

Dynamic light scattering identified high molecular weight species withboth A56P and TP alpha-S (FIG. 2C). The hydrodynamic radius wasapproximately 100 nm, a value very similar to that observed witholigomers of wt and A30P alpha-S (FIG. 2C and FIG. 8). Measurement ofmonomer consumption throughout the aggregation by 1D ¹H NMR spectroscopyrevealed that the oligomeric intermediates formed by A56P and TP alpha-Sconstitute a 6% and 2% fraction of the protein mixture after 50 hours,and a 40% and 4% fraction after 160 hours respectively (FIG. 2B). Amixture of oligomeric and monomeric TP alpha-S, which was obtained afterfive days of aggregation of TP alpha-S at 37° C. and 0.2 mM proteinconcentration, was able to seed fibril formation of monomeric wt alpha-S(FIG. 2D). In contrast, addition of the same concentration of purelymonomeric TP alpha-S did not accelerate aggregation of wt alpha-S. Inaddition, the mixture of oligomeric and monomeric TP alpha-S wasrecognized by the conformation-specific antibody A11 (FIG. 2E), whichdetects a variety of toxic amyloid oligomers. Notably, the TP oligomerswere not resistant to sodium dodecyl sulfate (data not shown).

Increasing the concentration to 0.8 mM significantly accelerated therate and amount of aggregation and amyloid formation of all alpha-Svariants (FIG. 2G). At 0.8 mM protein concentration, wt and A30P alpha-Shad a distinct lag phase of about 9-12 hours, whereas ThT reactivity ofA53T rose from the beginning (inset in FIG. 2G). On the other hand, A56Pstarted to form ThioT-positive fibrils after about 72 hours and TPalpha-S displayed a clear but very slow rising ThioT signal only afterabout 5 days of incubation (FIG. 2G). Surprisingly, the strongest ThioTsignal after 5 days of incubation was observed for A30P alpha-S,followed by wt and A53T alpha-S. This is most likely caused by the verygel-like behaviour of the aggregated wt, A30P and in particular A53Talpha-S sample that interfered with ThioT binding. The samples of A56Pand TP alpha-S were much more fluid, indicating that a smaller amount offibrils was formed. In addition, A56P and TP alpha-S had stronglyreduced fibril elongation rates. Whereas for wt, A30P and A53T alpha-Sit took about 20 hours to reach the saturating ThioT signal from end ofthe lag phase, this time was increased to about 60 hours in case of A56Palpha-S. With TP alpha-S a saturating ThioT signal could not be reachedwithin the experimental time, indicating that it has an extremely slowfibril elongation rate (FIG. 2G).

Electron microscopy of wt, A30P, A53T and A56P alpha-S samples after 6days of incubation (protein concentration of 0.8 mM) revealed a highnumber of fibrils of about 8 nm in diameter and various lengths butwithout clearly observable oligomeric species. In case of TP alpha-S,the fibrils were significantly lower in number, longer and frequentlyassociated with oligomers of various shapes and sizes (FIG. 20 and FIG.17A). Atomic force microscopy of the TP alpha-S sample showed a similarpicture, with the presence of fibrils of about 8 nm in diameter, andoligomers of 20-100 nm in diameter (FIG. 17B).

The aggregation process of the alpha-S variants was further investigatedby dynamic light scattering and electron microscopy. Dynamic lightscattering revealed the formation of soluble oligomers with ahydrodynamic radius of approximately 80-180 nm after six hours ofincubation in the aggregation assay employing protein concentrations of0.1 mM (FIG. 8). In the same assay, a heterogeneous distribution oflarger species was observed for all alpha-S variants after 12 hours ofincubation by electron microscopy (FIG. 8).

In addition, DLS was used to study the soluble oligomers of alpha-S,which were formed after 11 days of incubation. At protein concentrationsof 0.8 mM, fibrils were observed for all alpha-S variants (see above andFIG. 17A). The fibrillar material was separated from soluble oligomersby centrifugation at 14,000 rpm for 30 minutes and careful pipetting ofthe upper 50% of the supernatant. DLS measurements of the supernatantsamples showed quite different scattering patterns for the differentalpha-S variants. The smallest scattering intensity was observed for wtalpha-S (FIG. 17C). A30P and A53T alpha-S had very similar scatteringintensities, which were slightly larger than that of wt alpha-S, and forA56P alpha-S the scattering intensity was further increased. The mostdramatic increase, however, was seen for TP alpha-S, for which thescattering intensity of the supernatant sample was an order of magnitudehigher than in case of the wt protein (FIG. 17C) and mostly caused by140-170 nm oligomeric species. The UV absorbance spectrum of thesupernatant showed a very similar trend for the alpha-S variants (FIG.17D). The combined EM, AFM, DLS and UV data indicate that A56P and inparticular TP alpha-S have an impaired ability to form amyloid fibrils,but soluble oligomers accumulate in later stages of the aggregation.

The late-stage aggregates of A56P and TP alpha-S were characterized atsingle residue resolution by solid-state NMR (ssNMR) spectroscopy (FIG.10). For both A56P and TP alpha-S, magnetization transfer from water tothe protein proceeded with the same rate suggesting that the relativewater-accessible surface in these fibrils was similar to that of fibrilsfrom wt protein (FIG. 10A). Assuming a cylindrical (proto)fibril model,we estimated fibril diameters of about 60 A for all three cases.However, cross peak signals in sequential (¹³C,¹³C) correlationexperiments conducted on A56P alpha-S were absent around the mutationsite (e.g. Y39, S42, T54 and A56) but were identified for the otherthree beta-strand segments previously seen for the wt, A30P and A53Tprotein (Heise et al., 2005) (FIG. 10B). In addition, we detectedalterations in chemical shifts for residues including T75, Q79, I88, andE83, indicative of a perturbed beta-strand structure. These findingspoint to a reduced beta-sheet content in late stage aggregates of A56Palpha-S compared to wt, A30P and A53T alpha-S. Concomitantly, wedetected in ssNMR experiments probing mobile A56P fibril segments anenhanced contribution from residue types such as threonine, which arefound in the residue stretch 22-93 (FIG. 10C). For TP alpha-S, a furthersignificant reduction of cross-peak correlations was detected (FIG. 10D)under experimental conditions comparable to A56P, consistent withstructural alterations or increased dynamics/disorder in the last twobeta-strands (FIG. 10E).

Collectively, these results indicate that there were only subtle changeswith respect to the biophysical properties of mutant versus wild typealpha-S protein with the exception that the aggregation process wasdrastically slowed down.

Example 3

In Vivo Fibril Formation

Human Embriyonic Kidney (HEK) Cell Cultures

Cell Culture

HEK293 cells were cultured in Dulbecco's MEM (PAN-Biotech, Aidenbach,Germany) with 10% fetal calf serum and 1% penicillin-streptomycin. Cellswere transiently transfected using Metafectene (Biontex Laboratories,Martinsried, Germany), following the manufacturer instructions. Forimaging, cells were grown on poly-L-lysine (Sigma, Munich, Germany)coated glass coverslips and used 24 h after transfection. For stainingof chromatin and F-actin, cells where submerged in PBS with 0.5 μg/ml ofHoechst 33258 (Invitrogen) and 1:500 of Alexa-568 conjugated phalloidin(Invitrogen) for 15 min at room temperature and washed 3 times in PBS.Coverslips were mounted on glass slides using mounting medium consistingof 24% w/v Glycerol, 0.1 M Tris-base pH 8.5, 9.6% w/v Mowiol 4.88(Calbiochem, Darmstadt, Germany) and 2.5% w/v of DABCO (Sigma).

Imaging

Imaging at 24 h was performed at room temperature using an invertedfluorescence microscope (DMI6000B, Leica Microsystems, Bensheim,Germany) with a 63× dry objective (HCX PL FLUOTAR, N.A. 0.7) and a LeicaFX350 Camera. For each genotype, 200-300 cells per coverslip from from4-5 independent experiments were classified manually based on their EGFPdistribution as either “homogenous”, “with a single aggresome”, “withmany aggregates” or “preapoptotic”. For statistical analysis, One-WayANOVA was performed with GraphPad Prism 4.00 (GraphPad Software, SanDiego, USA). P values were derived from Dunnett's posttests. Allcomparisons were made against the control PDZ-EGFP alone. Bars depictedin the graphs represent mean±standard error of the mean.

Quantitative rtPCR.

Total RNA was isolated from HEK293 cells 24 h after transfection usingthe RNeasy Mini Kit (Quiagen, Hilden, Germany). RNA was digested by RQ1RNase Free DNase (Promega, Mannheim, Germany) and protected againstRNases by adding 20 U of RNase Inhibitor RNasin (Promega, Mannheim,Germany). 2.5 mg of total RNA was used for reverse transcriptase PCR(M-MLV; Promega, Mannheim, Germany). cDNA was diluted 1:50 and real-timereaction samples were prepared using ABsolute QPCR SYBR Green (ABgene,Hamburg, Germany), according to the manufacturer instructions. Real-timePCR was performed in a Stratagene Mx3000P Realtime device (Stratagene,La Jolla, Calif.). Primers for detection of a-synuclein were Fw_(—)5′CAGGGTGTGGCAGAAGCAGC3′ (SEQ ID NO: 4) and Rv_(—)5′CTGCTGTCACACCCGTCACC3′(SEQ ID NO: 5). Eucariotic 18s ribosomal mRNA was chosen as referencegene and quantification calculated using the comparative 2-ΔΔCt method.Water and pEGFP-N1 transfected cells were used as negative controls.Three independent experiments (n=3) were performed, each withtriplicates. Significance of expression differences, were tested usingone-way ANOVA, which was not significant.

Western Blot.

Cells were plated in 6 well plates at equal density and transfected thenext day. 24 h after transfection, cells were harvested in phosphatebuffered saline (PBS), centrifuged and resuspended in 100 μl of lysisbuffer: PBS with 1% TritonX and protease inhibitor cocktail (Pierce,Rockford, Ill., USA). Lysates were cleared by centrifugation (15,000 g,20 min, 4° C.) and the supernatant transferred to new tubes. 10 μl ofeach were separated by SDS-PAGE. Primary monoclonal antibody againstalpha-S was used over night at 1:1000 and at 4° C. (BD TransductionLaboratories, Cat. #610786). After incubation with the secondaryantibody and visualization, the membrane was washed 3 times 20 min withstripping buffer (0.2 M Glycin, 0.5 M NaCl, pH 2.80) and incubated overnight with antibody against GFP (polyclonal; Santa Cruz #SC 8334). Thesecondary antibodies for both primaries (GE Healthcare, #NXA931 &#NA934V) were coupled to horseradish-peroxidase (1:10000) and visualizedindependently by chemiluminescence (Alphalmager, Alphalnnotech, SanLeandro, Calif.). Quantification of aS signal was normalized against theEGFP signal of the same sample. This is a better expression controlsince EGFP is in the same vector but under a second CMV promotor).Beta-actin levels were equivalent among the different constructs, butnormalizing with beta-actin would control for transfection efficiency,and not for protein expression levels. Tree independent experiments(cells plated, transfections and western blots) were performed.

The in vivo formation of insoluble aggregates in human embryonic kidney(HEK) cells was tested after expression of fluorescently labelled A30P,A53T, A56P and TP alpha-S, having a six-amino acid PDZ binding motif forrecognition by enhanced green fluorescent protein (EGFP). Equalexpression levels of all alpha-S variants were verified by quantitativePCR and Western blot (FIG. 18). A second, independent cassette expresseda fusion protein of EGFP and the corresponding PDZ domain (PDZ-EGFP),thus non-covalently labelling alpha-S variants with EGFP. We classifiedcells based on their EGFP fluorescence to determine the frequency ofcells with aggregates and the fraction of preapoptotic cells (seeabove). Significantly more cells transfected with A30P or A53T alpha-Sformed aggregates (as visualized by EGFP fluorescence) as compared tocells expressing the control protein PDZ-EGFP alone (FIGS. 18A and 18B;FIG. 2F, clear bars). In contrast, expression of the design mutants A56Pand TP alpha-S mutants did not induce aggregates in more cells thanbackground (EGFP-PDZ alone). Thus, A56P and TP alpha-S fulfilled theirdesign principle, i.e. they show strongly impaired aggregation both invitro and in living cells. Expression of the genetic mutants A30P andA53T alpha-S resulted in more cells with aggregates and a higherfraction of preapototic cells than control. (FIG. 18A; FIG. 2F, hatchedbars). In contrast, expression of A56P and TP alpha-S resulted intoxicity comparable to that observed for A53T alpha-S, despite theobservation that the occurrence of aggregates was at background levels(PDZ-EGFP alone). From the induction of toxicity but not aggregates bythe design mutants A56P and TP alpha-S we conclude that cellulartoxicity in response to alpha-S expression does not require theformation of visible alpha-S aggregates. However, differences intoxicity between A56P and TP alpha-S that are related to aggregationcannot be revealed in this system, as already the single A56P mutationhad a dominant effect on aggregation.

Example 4

Intervertebrate Animal Models

C. elegans Experiments

Expression Constructs

As described previously (Pitman et al., supra), a 719 bp dat-1 promoterfragment was PCR amplified and cloned upstream of the start ATG ofenhanced gfp in the C. elegans expression vector pPD115.62 (myo-3::gfp;kindly provided by A. Fire) in order to express alpha-S in dopaminergicneurons, replacing the myo-3 promoter creating Pdat-1::gfp. Subsequentlyalpha-S and its mutant variant were also PCR amplified and cloned as aNdeI/HindIII fragment into the Pdat-1::gfp vector replacing GFP. Tocreate Pdat-1::mCherry, the gfp coding sequence of Pdat-1::gfp wasexchanged with that of the red fluorescent protein variant mCherry. Toanalyze aggregation alpha-S—mYFP citrine fusion proteins werespecifically expressed in muscle cells under the control of the myo-3promoter of pPD115.62. Wt and TP alpha-S were PCR amplified without stopcodon for C-terminal fusion and cloned along with mYFP citrine intopPD115.62 replacing GFP, resulting in Pmyo3::αS-YFP. All constructs wereverified by sequencing.A11 constructs were verified by sequencing.

Transgenic Animals

C. elegans strains were cultured as described previously (Brenner, S.,supra) and kept at 20° C. if not otherwise stated. To create transgenicanimals expressing alpha-S or its mutants in dopaminergic neurons thegonads of young adult wild type N2 hermaphrodites were injected with aplasmid mix of Pdat-1::alpha-syn (60 ng/μl) and Pdat-1::mCherry (40ng/μl) as co-injection marker. The concentration of the alpha-Sexpression constructs were chosen such that wild type alpha-S expressionat this given concentration shows only a weak phenotype. Theconcentration of all other alpha-S expression constructs was keptconstant accordingly. To express mYFP citrine tagged alpha-S variants inbody wall and sex muscles a plasmid mix containing Pmyo3::αS-mYFP (40ng/μl) and the coinjection markers pRF4 (rol-6(su1006sd); 40 ng/μl) andPttx3::gfp (10 ng/μl) were injected. To allow comparable expressionlevels, the injection mix was always adjusted to a total DNAconcentration of 100 ng/μl by adding pBlueScript SKII (Stratagene). Onlytransgenic lines showing highly uniform expression were selected andsimilar levels of alpha-S expression were confirmed by RT-PCR andWestern Blot. Alpha-S was detected using a polyclonal rabbit αS antibody(Anaspec). All blots were normalized against alpha-tubulin usingmonoclonal Ab 12G10 (DSHB). To image alpha-S-mYFP aggregation in musclecells 10 day old trangenic animals were anesthetised and imaged usingthe UltraviewVOX spinning disk microscope (PerkinElmer). At least twoindependent strains per alpha-S variant were imaged. Vulva muscles werescored positive if at least one fibrilar aggregate was visible.

Microscopy and Behavioral Analysis

C. elegans contains eight dopaminergic neurons which are involved infood sensation. These neurons have been widely used as an accepted modelsystem to mimic Parkinson related phenotypes in C. elegans. As in thehuman system exposure of the worm to 6-hydroxydopamine (6-OHDA) or MPTPresults in a specific degeneration of dopaminergic neurons andassociated alterations in dopamine controlled behaviors. Furthermore,this toxicity is dependent on the presence of the dopamine transporterDAT-1 as no degeneration is observed in dat-1 mutant animals or ifdopamine transporter inhibitors are used. Thus C. elegans can also beused to test and find neuroprotective compounds (Marvanova & NicholsJournal of Molecular Neuroscience 31, 127-137 (2007)). This exemplifiesthat the dopaminergic system of C. elegans is highly similar tomammalian system and can therefore be used as a model to assay theneuronal physiology linked to Parkinson's disease (PD). Accordingly,expression of alpha-S or its mutant versions linked to PD specificallyin dopaminergic neurons have been shown to cause neuronal toxicity anddegeneration (Pitman et al., supra; Shaw et al. Science 287, 1834-1837(2000)). Interestingly, in C. elegans familial PD linked A30P alpha-Sand A53T alpha-S exhibit an increased neuronal toxicity as compared towild type alpha-S (Pitman et al., supra).

Routinely, transgenic animals were imaged or assayed four days afterreaching adulthood at least three independent strains per transgene weretested. To image dopaminergic neurons, transgenic animals wereanesthetized by 50 mM sodium azide in M9 buffer and mounted on a 2%agarose pad. RFP positive dopaminergic neurons were visualized using aLeica SP2 confocal microscope system. Neurite defects were scoredpositive if one or more dendritic processes out of four had degenerated.For each transgenic strain at least 75-80 animals were tested.

As a response to the presence of food wild type animals slow down theirmovement and reduce their area restricted searching behavior in order tofeed more efficiently. This dopamine-controlled behavior is absent whendopaminergic neurons are ablated or not functional. Therefore, thisbehavior allows to directly assess the functional integrity ofdopaminergic neurons in C. elegans. For behavioral analysis wellfedadult animals were transferred to the center of an assay plate with orwithout food as described previously (Brenner, supra). After an initialtime of adjustment for 5 min the movement was assayed by counting bodybends over a one min interval with three repetitions per animal. Theslowing rate was calculated and defined as the percentage of locomotionon food as compared to the locomotion on plates without food. For eachtransgenic strain at least 35-50 animals were tested in double blindfashion. Each trail was repeated three times.

Drosophila Experiments

Generation of Transgenic Flies

The site-specific recombination system based on φC31 integrase was usedto generate transgenic flies (Sawin et al. Neuron 26, 619-631 (2000)).The targeting constructs were prepared by cloning the cDNAs of alpha-Svariants into the GAL4-responsive pUAST expression vector containingattB site (attachment site B). The resulting plasmids were then injectedinto the fly embryos, which are double homozygous for both attP(attachment site P) site and germ-linespecific φC31 integrase. Thegenomic location of the attP landing site used for integration wasmapped to the 3R-86Fb position in the genome (ZH φX-86Fb line) (Sawin etal., supra). All the site-specific insertions were verified by singlefly PCR using the primer pairs: 5′ACT GAA ATC TGC CAA GAA GTA 3′ (SEQ IDNO: 2) and 5′GCA AGA AAG TAT ATC TCT ATG ACC 3′ (SEQ ID NO: 3). In orderto compare the ddc-Gal4 driven protein expression levels in transgenicanimals expressing different variants of alpha-S, SDS-PAGE andsubsequent western blotting were performed from fly head extracts asdescribed previously (Edery et al. Proc Natl Acad Sci US A 91, 2260-4(1994)).

Immunohistochemistry.

Whole-mount adult fly brains from the 28-30 day old animals wereprepared and immuno-stained. Rabbit anti-tyrosine hydroxylase (TH)(1:150; Chemicon International, Temecula, Calif.) was used to positivelystain the DA neurons, and Mouse anti-nc82 (1: 200; Developmental StudiesHybridoma Bank, University of Iowa, Iowa City, Iowa) was used as acounter stain. From the confocal sections of fly brains of differentgenotypes, DM and DL clusters of DA neurons were defined and counted byusing the ImageJ64 software (National Institutes of Health, Maryland,USA) (10-15 brains per genotype; two independent experiments).

Behavioral Analysis

Longevity Assay

Flies expressing alpha-S variants and control animals expressing lac Zwere collected and maintained under LD 12:12 at 25° C. with constanthumidity and population density per vial. Flies were transferred to thefresh food vials and scored for survival every 5 days. Survival curveswere calculated and plotted using Kaplan-Meier statistics, anddifferences between them were analysed by using the log rank method(GraphPad Prism software, San Diego, USA).

Sleep Assay

Fly embryos of different genotypes were collected in 2 hour windowperiods, and were grown under LD 12:12 at 25° C. before the eclosion.Males were collected from the progeny and aged with equal populationdensity under LD 12:12 at 25° C. After 25-30 days, locomotor activity ofthe aged flies was recorded in LD by the Drosophila Activity Monitoring(DAM) system (Trikinetics, Waltham, Mass.) as described in (Hendricks etal. Nat Neurosci 4, 1108-15 (2001); Hendricks et al. Neuron 25, 129-38(2000)). Sleep was measured as bouts of 5 min of inactivity, using amoving window of 1 min intervals. Average bout length (ABL) wascalculated from the sum of sleep bouts of all lengths (in minutes)divided by the total number of sleep bouts.

Climbing Assay

Flies expressing different alpha-S variants were placed in an apparatuscontaining a bottom vial and an inverted upper vial. They were assayedfor their ability to reach upper vial from the bottom vial in twentyseconds. During the assay, to avoid photic effects from outsideenvironment, both vials have been encased in black cases. Since fliesgenerally get attracted towards light, a light source at the top ofupper vial with the help of two light emitting diodes was also provided.This type of set up provides a directionality and motivation for theflies to climb up.

Results

To further explore the consequences of delayed alpha-S aggregateformation in living organisms and to elucidate its functionalconsequences, the impact of the biophysically characterized alpha-Smutants on various behavioural aspects related to PD in model organismssuch as locomotor activitiy of fly Drosophila melanogaster and thenematode C. elegans was tested. Expression levels of all alpha-Svariants in a specific model system were comparable (FIGS. 18, 19 and23).

C. elegans

When expressed in muscle cells of C. elegans, wt alpha-S had been shownto form aggregates in an age dependent manner. To demonstrate that theTP variant of alpha-S has a strongly impaired ability to form insolubleaggregates in vivo—as has been shown in vitro—we expressed wt and TPalpha-S as a fusion to monomeric YFP in body wall and sex muscles of C.elegans and followed its aggregation with time. As reported previouslyaggregates of wt alpha-S-mYFP are first detected in six-day old adultmuscles and at day 10 large fibrillar aggregates were visible in mostmuscles (86% and 75% in 25 animals analyzed in each of two independentstains, respectively) (FIG. 19A). In contrast, at day six no TPalpha-S-mYFP aggregates could be detected in any of the transgenicstrains and even at day 10 only rare, small TP alpha-S-mYFP aggregateswere visible in a few muscle cells (4% and 8% in 26 animals analyzed ineach of two independent stains, respectively) (FIG. 19B). Thealpha-S-mYFP expression levels were comparable as judged by Western blotanalysis and even slightly higher for the TP strains used (FIG. 19C).Therefore, we conclude that, like in vitro, the TP mutations stronglyimpair fibril formation of alpha-S in vivo.

A hallmark of PD is the progressive loss of dopaminergic neurons inpatients. Dopaminergic neurons in C. elegans have been successfully usedas a model system to assay the toxicity of alpha-S mutants associatedwith familial PD (Nass & Blakely Annu Rev Pharmacol Toxicol 43, 521-544(2003)). The six dopaminergic neurons in the head of C. elegans arecleary visible and morphologically invariant from animal to animal,enabling reliable scoring of morphological defects (Nass & Blakely,supra). To assay alpha-S induced neuronal toxicity, transgenic strainswere generated expressing the different alpha-S variants exclusively indopaminergic neurons of C. elegans. As these neurons are dispensable andare not required for viability, their alpha-S induced degeneration canbe studied without affecting the animal's fitness. For each expressedalpha-S variant, the morphology of dopaminergic neurons in the head inmultiple independent transgenic strains was analyzed. Two representativelines each are shown in FIG. 4B. Transgenic strains overexpressing thegenetic mutants A30P (45±5% and 42±4%) and A53T (36±6% and 48±5%)alpha-S developed more neurite defects than control animals expressingwt alpha-S (13±5% and 8±3%) (FIG. 4B). More pronouncedneurodegeneration, however, was observed for C. elegans strainsexpressing the designed alpha-S variants A56P (63±3% and 68±3%) and TP(88±2% and 81±3%) (FIG. 4B). Importantly the A56P alpha-S mutant causedmore severe neurite defects than the A30P alpha-S mutant. In bothalpha-S variants, alanine is replaced by proline within the alpha-Sdomain that converts from an unfolded conformation in the solublemonomer to an alpha-helical structure by interaction with membranemimetics. Within this domain, A30 is not part of the rigidbeta-structure of alpha-S fibrils whereas A56P carries the replacementright in the center of the second beta-strand (FIG. 1A) (Heise et al.,supra). The alpha-S expression levels were similar in all strainsstudied as shown by Western blot analysis using an alpha-S-specificantibody (FIG. 23). However, the degeneration was not restricted todopaminergic neurons. When alpha-S was expressed under the control of apan-neuronal promoter degeneration of other neurons was visible leadingto sick animals (data not shown).

In response to the presence of food, C. elegans worms slow down movementand reduce the area-restricted searching behaviour. This behaviourdepends on dopaminergic neurotransmission and is absent whendopaminergic neurons are ablated or not functional (Sawin et al.,supra). Transgenic worms expressing the A56P or TP alpha-S variant indopaminergic neurons showed a strong impairment of this dopamine(DA)-dependent behaviour. This impairment was strongly enhanced ascompared to animals expressing the A30P or A53T mutant or wt alpha-S(FIG. 3A).

D. melanogaster

Next wt alpha-S, A53T, A56P or TP alpha-S mutant proteins were expressedin Drosophila. To ensure comparable expression of the different alpha-Svariants, the corresponding transgenes were targeted to the same genomiclocation by using the φ-C31 based site-specific recombination system(FIG. 11).

To assess the impact of the alpha-S variants on dopaminergic neurons inDrosophila, we immunostained whole-mount brains from flies at 2 and 29day posteclosion with an antibody against tyrosine hydroxylase, whichspecifically identifies these neurons (FIG. 4C). In young fliesoverexpressing wt, A53T, A56P and TP alpha-S under control of thepan-neuronal driver elav-Gal4 the number of neurons in the dorsomedial(DM) and dorsolateral (DL) cluster of the brain was not altered whencompared to the LacZ control (FIG. 4D). At day 29, however, adult fliesexpressing A53T and A56P alpha-S demonstrated a marked loss oftyrosine-hydroxylase-positive cells in both clusters (FIG. 4D). An evenmore pronounced reduction in the number of dopaminergic neurons wasobserved in flies expressing TP alpha-S, in particular in the DM cluster(FIGS. 4C,D).

Using the pan-neuronal driver elav-Gal4, flies were assayed for motordefects using a climbing assay which addresses the combined geotacticand phototactic response of flies. The loss of the climbing response hasbeen used to monitor aging-related changes in Drosophila and to revealbehavioral manifestations of nervous system dysfunction in alpha-Stransgenic flies. The climbing abilities of 25-30 day old fliesexpressing wt alpha-S (or A30P alpha-S according to initial tests) werecomparable to those of the LacZ control flies (FIG. 3B). In contrast,flies expressing the genetic mutant A53T alpha-S or theaggregation-impaired design mutant A56P alpha-S showed a reducedclimbing ability. In agreement with the lowest number of dopaminergicneurons (FIG. 4D), adult flies expressing TP alpha-S were most stronglyimpaired (FIG. 3B).

To further explore the effects of wt alpha-S and alpha-S mutants whendirectly expressed in the dopamine producing target cells, a driver linewas used that contains the promoter for the DOPA decarboxylase gene(ddc-Ga14) which then allows transgene expression in a subset of neuronsincluding dopaminergic neurons.

It was found that overexpression of mutant alpha-S did not affect thesurvival rate within the first 33 days, after that, however, fliesoverexpressing A56P alpha-S, and in particular TP alpha-S, had a highermorbidity resulting in an average reduction of the life span byapproximately 10 days when compared to wt alpha-S flies (FIG. 3C andTables 2 and 3). Furthermore, 25-30 day old flies expressing thedesigned mutants showed aberrant sleep patterns (FIG. 3D,E), which werestrongest for flies expressing the A56P and TP alpha-S mutants,affecting both the sleep profiles and the average lengths of sleep bouts(FIG. 3D,E and FIGS. 12, 13). It is interesting to note that DA neuronsin Drosophila innervate the mushroom body, a brain area involved insleep regulation (Pitman et al., supra), and common features have beensuggested between sleep states in insects and mammals (Shaw et al.,supra)). Thus, flies expressing toxic variants of alpha-S might providea means to investigate the mechanism underlying sleep impairment bygenetic and pharmaceutical tools that in turn can be useful fordeveloping PD therapy.

TABLE 2 Comparison of Survival Curves wt alpha-S and A56P alpha-SLogrank Test Chi square 5.270 df 1 P value 0.0217 P value summary * Arethe survival curves Yes sig different? Median survival Data 1:a-Syn-wt48.00 Data 1:a-Syn-A56P 42.00 Ratio 1.143 95% CI of ratio 0.2754 to2.010 Hazard Ratio Ratio 0.8769 95% CI of ratio 0.6766 to 0.9697

TABLE 3 Comparison of Survival Curves wt alpha-S and TP alpha-S LogrankTest Chi square 47.74 df 1 P value P < 0.0001 P value summary *** Arethe survival curves Yes sig different? Median survival Data 1:a-syn(wt)48.00 Data 1:a-syn(triple) 42.00 Ratio 1.143 95% CI of ratio 0.2733 to2.012 Hazard Ratio Ratio 0.6946 95% CI of ratio 0.4266 to 0.6217

Taken together the data demonstrate that over-expression of alpha-Svariants, which delay fibril formation but allow oligomer formation,causes increased neurotoxicity in established model systems for PD:increasing impairment to form fibrils is consistently correlated withincreasing neurodegeneration (wt˜A30P<A56P<TP). The genetic mutant A53Talpha-S has a neurotoxicity comparable to that of A56P alpha-S in thethree model systems, but does not allow a conclusion about theimportance of a certain aggregate species for neurotoxicity as A53Talpha-S forms both oligomers and amyloid fibrils more rapidly. Theseresults indicate that delayed fibril formation by the structure-baseddesign mutants of alpha-S causes functional impairments in bothDrosophila and C. elegans, that can be attributed to dopaminergicdysfunction. They alter mobility of flies and worms, affect the sleepingbehaviour of flies and reduce their lifespan.

Example 5

Mammalian Neurons

Primary Neuronal Cultures

AAV-1/2 mosaic serotype viral vectors were prepared essentially asdescribed (Nass & Blakely, supra). Their genomes consisted of AAV-2ITRs, human synapsin-1 gene promoter driving expression of alpha-Svariants, WPRE for enhanced mRNA stability and bovine growth hormonepolyadenylation site.

Primary cortical and midbrain neurons were prepared from rat embryos atE18 or E16, respectively. Neurons were plated in 96 well plates for WSTassay and tyrosine hydroxylase (TH) immunocytochemistry and in 24 wellplates for NeuN immunocytochemistry. Neurons were transduced by AAVvectors at day 3 in vitro (DIV 3) and were analysed at DIV 10. WSTassay, measuring mitochondrial dehydrogenase activity, was performedaccording to the protocol of the manufacturer (Roche Diagnostics).Immunocytochemistry was performed with anti-NeuN (Chemicon) and anti-THantibodies (Advanced Immunochemicals Inc.) detected by Cy3 coupledsecondary antibody. Cell counts were performed in at least 5 randomlyselected fields per well and in at least 6 wells each of at least twoindependent replicates of respective transduction by AxioVision software(Zeiss).

To perform statistical analysis, respective groups were tested byLevene's test for equality of variances in order to confirm that One-WayANOVA could be performed, for which Student-Newman-Keuls test for allpair wise comparisons was used. Data are presented as mean±standarderror of the mean (SEM).

Results

We further investigated toxic effects of the alpha-S mutants uponexpression in cultured mammalian neurons. Rat primary cortical neuronswere transduced with Adeno-associated-virus (AAV) (Kugler et al. Am JHum Genet 80, 291-297 (2007)) expressing wt or mutant alpha-S. Transgeneexpression was absolutely neuron-restricted and transduction efficacywas in the range of 95% as evidenced by EGFP fluorescence. Using awater-soluble tetrazolium salt (WST) colorimeteric assay formitochondrial dehydrogenase activity, we found that A56P alpha-S wasmore toxic than A53T alpha-S (which did not show significant differencesto wt and A30P alpha-S, data not shown) and control neurons expressingEGFP (FIG. 4A). Furthermore, the TP alpha-S variant was significantlymore cytotoxic than the A56P mutant (FIG. 4A). Since the WSTcolorimeteric assay estimates not only the mitochondrial capacity toproduce reduced equivalents but also the decline of mitochondrialactivity due to diminished cell numbers, numbers of neurons by neuronalnuclei (NeuN) immunocytochemistry were also counted after expression ofA53T, A56P and TP alpha-S variants as well as EGFP control protein. Itwas found that the TP mutant was again the most cytotoxic alpha-Sspecies (FIG. 4A). However, while both A53T and A56P over-expressionsignificantly reduced cortical neuron numbers as compared with EGFPover-expression, their effect was not statistically different from eachother as in the WST assay, indicating that A56P might exert more subtleneurotoxic effects not directly leading to neurodegeneration as observedwith the TP mutant. Finally, it was explored whether and which of thealpha-S mutants exerts a specific neurotoxic effect in dopaminergicneurons. Expression of A53T, A56P and TP alpha-S resulted in a lowermitochondrial dehydrogenase activity and a reduced number of survivingneurons than wt and A30P alpha-S and control neurons expressing EGFP(FIG. 4A). Similarly, in primary midbrain cultures the number ofdopaminergic neurons was decreasing in the orderEGFP≈wt≈A30P>A53T˜A56P>TP (FIG. 4A). It is important to note that thegenetic mutant A30P, in which the single proline mutation occurs outsidethe core of alpha-S fibrils, had a similar effect as wt and A53T alpha-Sin cultured mammalian neurons (data not shown). This observation isconsistent with a failure to detect neurodegeneration in transgenic miceoverexpressing A30P alpha-S (Lee et al., supra).

A strong correlation between delayed formation of and lack of beta-sheetcontent in fibrils of alpha-S variants and the strength of PD-relatedbehavioural effects of the alpha-S variants was found when expressed inanimal models such as Drosophila and C. elegans and with increasedneurotoxic effects on dopaminergic neurons of both C. elegans andmammals. These findings highlight the importance of pre-fibrillar,soluble alpha-S species in the pathogenesis and progression of PD andother neurodegenerative disorders collectively referred to assynucleinopathies and suggest that the inhibition of alpha-S mutants toform beta-sheets correlates with toxicity. Furthermore, thecorresponding biological effects including the degree of toxicity in thedifferent PD models used here suggest that structure-based designmutants of alpha-S provide a powerful tool for the identification ofpotential therapeutic compounds. In particular, the strongneurodegeneration exerted by TP alpha-S raises the possibility that celldeath and neurodegeneration will be observed early on in rodent modelsof PD.

Example 6

Design of Alpha-S Variants

A design of alpha-S variants that aims at the production of toxicspecies should keep the structural and functional properties of therespective multimers nearly constant. The design was based on theconformational properties of the alpha-S monomer in solution(Bertoncini, C. W., et al. Proc Natl Acad Sci USA 102, 1430-1435(2005)). V118 and M127A are hydrophobic residues that are located in thehighly negatively charged C-terminal domain of alpha-S, which folds backonto the hydrophobic NAC region that is essential for aggregation ofalpha-S into amyloid fibrils. To interfere with the intramolecularinteraction between the C-terminal domain and the NAC region of alpha-S,which shields alpha-S from self association, V118 and M127 were replacedby alanine.

Cloning, Expression, and Purification of Alpha-S Variants

The following alpha-S variants were generated as described in Example 1:the single mutants M127A, V118A and the double mutant V118A+M127A. Basedon the intramolecular interactions observed in monomeric alpha-S, thesemutations are expected to cause a destabilization of the folding nucleusof alpha-S and lead to increased self-association.

NMR Spectroscopy

NMR samples contained ˜0.2 mM 15N-labelled mutant alpha-S in 50 mMNa-phosphate buffer, 100 mM NaCl at pH 7.4 and 90% H₂O/10% D₂O. Theexperiments were recorded on a Bruker Avance 600 MHz NMR spectrometer.The temperature was set to 15° C. unless otherwise stated. Dataprocessing was performed using the software packages Topspin (Bruker)and Sparky (Goddard, T. D., Kneller, D. G., University of California,San Francisco). For chemical shift analysis, 1H-15N Heteronuclear SingleQuantum Coherene (HSQC) 2D spectra were recorded.

NMR spectroscopy showed that substitution of M127 and V118 by alanine orE or D does not induce rigid secondary or tertiary structure. Instead,the small chemical shift dispersion of NMR signals indicates that thevariant alpha-Syn remains highly dynamic and samples a large ensemble ofconformations.

C. elegans Experiments

The C. elegans experiments were carried out as described above inExample 4. FIG. 16 A clearly shows that worms expressing M127A alpha-Sor A30P+A56P+A76P alpha-S are statistically more likely to show neuritedefects in comparison to worms expressing either wild-type alpha-S orA30P alpha-S. This data is consistent to the results depicted in FIG. 16B, showing a decreased slowing rate for worms expressing M127A alpha-Sor A30P+A56P+A76P alpha-S in comparison to worms expressing eitherwild-type alpha-S or A30P alpha-S.

Yeast Experiments

Yeast cells were inoculated to an OD600 of 0.1 and incubated underalpha-S inducing conditions (SC-ura/Galactose). Total growth wasmeasured at OD600 after 16 h of incubation. Three independentmeasurements were performed. Toxicity of the alpha-S variants can bejudged by the growth impairment compared to vector control. FIG. 15shows a strong impaired growth for yeast cells expressing M127A alpha-Sand M127A+V118A alpha-S in comparison to the vector control, or cellsexpressing either wild-type alpha-S, A30P alpha-S, or A53T alpha-S.

Discussion

A better understanding of the relationship between the process ofalpha-S amyloid formation and disease progression in animal models forParkinson's disease is essential for understanding the molecular basisof neurodegeneration and the development of effective therapeuticstrategies to prevent and treat PD and other synucleinopathies. Wepresented a structure-based rational design of alpha-S mutants and theirbiophysical properties in vitro. The results establish that alpha-Smutants that cause reduced fibrillization and beta-structure formationand lead to the formation of increased amounts of soluble oligomers canbe predicted. We demonstrate that differences in the biophysicalproperties of these mutants translate into “predictable” changes inneuronal toxicity and behavioral defects in neuronal cell cultures andanimal models of synucleinopathies. The structure-based design mutantsprovide unique tools to dissect the relative contribution of oligomersand fibrils to alpha-S toxicity and establish the relationship betweenbiophysical properties of multimeric alpha-S species and their functionin different in vivo models.

The rational design of the alpha-S variants was based on the flexibilityof the alpha-S backbone in the monomeric state and the location ofbeta-strands in amyloid fibrils (Bertoncini et al., 2005, supra, Heiseet al., 2005). The genetic mutation A30P is located in a domain that isstatically disordered and not part of the core of amyloid fibrils ofalpha-S. We carried the alanine-to-proline replacement right into thecenter of the beta-strands of amyloid fibrils of alpha-S (FIG. 1A). Inagreement with the design principle, even the single point mutation A56Pstrongly reduced aggregation of alpha-S both in vitro (FIG. 2) and inliving cells (FIG. 18). Assuming that only drastic differences in therate of aggregation in vitro can be reliably transferred to the in vivosituation, the A56P mutation was complemented by the triple prolineA30P/A56P/A76P mutation, which shows impaired formation of insolubleaggregates in vitro (FIG. 2) and in vivo (FIG. 19). At the same time,however, both the A56P and the A30P/A56P/A76P alpha-S variant showed astrongly increased propensity to form soluble oligomers (FIG. 17). Incombination with wt alpha-S, the two structure-based design mutantsallowed a detailed study of the relationship between oligomerization,fibril formation and neurotoxicity in animal models for PD.

HEK cells, rat primary neurons, C. elegans and Drosophila thatover-express alpha-S are established models for PD. Here weover-expressed the wt protein, the genetic mutants A30P and A53T and thetwo structure-based design mutants A56P and TP alpha-S in all four ofthese model systems. The simultaneous use of four model systems wasmotivated by previous reports that over-expression of wt, geneticmutants and phosphorylation mimics of alpha-S induced different degreesof toxicity in different PD model systems. In contrast, expression ofthe structure-based design variants A56P and TP alpha-S caused increasedneurotoxicity in all four model systems: increasing impairment to formfibrils was consistently correlated with increasing neurodegeneration(wt A30P<A56P<TP) (FIGS. 3 and 4). This provides strong evidence for theimportance of soluble oligomers as the most toxic species in PD. Inagreement with the importance of soluble oligomers forneurodegeneration, other studies have suggested that aggregationintermediates are the pathologically relevant species in Alzheimer andHuntington disease.

A mutational strategy as employed in our study allows correlationsbetween biophysical properties observed for the mutated proteins invitro and functional deficits observed in vivo. In agreement with thedesign principle, the most dramatic effect observed for thestructure-based design variants of alpha-S was their impairedfibrillation but strongly enhanced formation of soluble oligomers. Inagreement with this design principle, our studies showed that the A56Pvariant of αS has an affinity for phospholipid vesicles that iscomparable and even slightly higher than A30P alpha-S (Table 1). Evenfor the triple-proline variant TP alpha-S an only slightly reducedvesicle-affinity (compared to A30P alpha-S) was observed, suggestingthat the alpha-S variants are flexible enough to efficiently bind tophospholipid vesicles. Despite the very similar vesicle affinities,however, only the A56P and A30P/A56P/A76P variant of alpha-S showed astrongly increased neurotoxicity, consistent with their higherpropensity to form soluble oligomers.

Our solid-state NMR data of late-stage aggregates of A56P and TP alpha-Sshowed that their morphology is similar to amyloid fibrils of wtalpha-S, however the molecular level structure is strongly changed. Adramatically diminished beta-sheet rich core was observed (FIG. 10),which suggests that soluble oligomers formed by the structure-baseddesign variants might also have a reduced ability to adoptbeta-structure. Importantly, neurotoxicity of variants of alpha-S(wt˜A30P<A56P<TP alpha-S) in the four model systems for PD was inverselycorrelated with the amount of beta-structure detected in insolublealpha-S aggregates (wt˜A30P>A56P>TP alpha-S) (FIGS. 2, 4, 18). Thusformation of rigid beta-structure might not to be as important forneurotoxicity as previously thought.

In conclusion, our combined biophysical and in vivo data revealed astrong correlation between enhanced formation of soluble oligomers andlack of beta-sheet content in fibrils of alpha-S variants, andneurotoxicity, the strength of PD-related behavioural effects andsurvival in four model systems for PD. This provides strong evidencethat structurally less stable aggregation intermediates of alpha-S arekey players in the pathogenesis and progression of PD and otherneurodegenerative disorders collectively referred to assynucleinopathies. The ability to engineer mutants that promote andstabilize specific toxic intermediates is essential not only forunderstanding the structural basis of alpha-S toxicity, but also fordeveloping diagnostic tools and imaging agents.

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US 2007/0192879

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The invention claimed is:
 1. A genetically engineered polynucleotideencoding an alpha-synuclein comprising at least 96% amino acid sequenceidentity to SEQ ID NO: 1, and having increased toxicity compared towild-type alpha-synuclein having the amino acid sequence of SEQ ID NO: 1wherein said toxicity is tested in a neuronal cell using an in vitrowater soluble tetrazolium (WST) assay, and wherein the alpha-synucleincomprises at least one amino acid substitution at the alanine atposition 56 (A56) and/or at the valine at position 118 (V118).
 2. Anexpression vector comprising the polynucleotide of claim
 1. 3. A cellcomprising the polynucleotide according to claim 1, wherein the cell isa yeast cell, an invertebrate cell, or a vertebrate cell.
 4. The cell ofclaim 3 wherein the invertebrate cell is a cell of C. elegans or a cellof D. melanogaster.
 5. The cell of claim 3 wherein the vertebrate cellis a mammalian cell.
 6. The cell of claim 5 wherein the mammalian cellis a mouse cell, a rat cell or a primate cell.
 7. The cell of claim 5wherein the mammalian cell is a non-human embryonic stem cell.
 8. Thecell of claim 5 wherein the mammalian cell is a dopaminergic neuronalcell.